U.S. patent number 6,194,181 [Application Number 09/248,910] was granted by the patent office on 2001-02-27 for fermentative preparation process for and crystal forms of cytostatics.
This patent grant is currently assigned to Novartis AG. Invention is credited to Hans Hofmann, Ernst Kusters, Marion Mahnke, Klaus Memmert, Michael Mutz, Frank Petersen, Thomas Schupp.
United States Patent |
6,194,181 |
Hofmann , et al. |
February 27, 2001 |
Fermentative preparation process for and crystal forms of
cytostatics
Abstract
The invention relates to a new process for concentrating
epothilones in culture media, a new process for the production of
epothilones, a new process for separating epothilones A and B and a
new strain obtained by mutagenesis for the production of
epothilones, as well as aspects related thereto. New crystal forms
of epothilone B are also described.
Inventors: |
Hofmann; Hans (Ettingen,
CH), Mahnke; Marion (Steinen, DE), Memmert;
Klaus (Lorrach, DE), Petersen; Frank (Weil am
Rhein, DE), Schupp; Thomas (Mohlin, CH),
Kusters; Ernst (Eschbach, DE), Mutz; Michael
(Freiburg, DE) |
Assignee: |
Novartis AG (Basel,
CH)
|
Family
ID: |
25684443 |
Appl.
No.: |
09/248,910 |
Filed: |
February 12, 1999 |
Foreign Application Priority Data
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|
|
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Feb 19, 1998 [CH] |
|
|
396/98 |
May 5, 1998 [CH] |
|
|
1007/98 |
|
Current U.S.
Class: |
435/118 |
Current CPC
Class: |
C12N
1/205 (20210501); A61P 35/00 (20180101); C07D
417/14 (20130101); A61P 35/04 (20180101); A61P
43/00 (20180101); C12P 17/181 (20130101); C12P
17/167 (20130101); C07D 493/04 (20130101); C07K
2299/00 (20130101); C12R 2001/01 (20210501) |
Current International
Class: |
C12P
17/16 (20060101); C07D 493/04 (20060101); C07D
493/00 (20060101); C12P 17/18 (20060101); C12P
017/16 () |
Field of
Search: |
;435/118 |
References Cited
[Referenced By]
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Foreign Patent Documents
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1222697 |
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41 38 042 |
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42 07 922 |
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WO |
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WO |
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WO |
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WO99/01124 |
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Jan 1999 |
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WO |
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WO 99/65913 |
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Dec 1999 |
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WO |
|
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.
Chemical Abstracts 98:124208s, Koho, JP 57,194,787, Nov. 30, 1982.
.
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.
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10, pp. 1017-1025 (1996). .
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.
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.
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|
Primary Examiner: Lilling; Herbert J.
Attorney, Agent or Firm: Lee; Michael U.
Claims
What is claimed is:
1. A process for producing epothilones in a culture medium for the
biotechnological preparation of these compounds, the process
comprising
(a) providing microorganisms which produce these compounds in the
medium, and
(b) adding a complex-forming component which is soluble in the
culture medium to the medium and forms complex with the
compounds.
2. A process according to claim 1 wherein said microorganisms are
myxobacteria.
3. A process according to claim 1, in which
(a) a culture medium is used for the biotechnological preparation
of epothilones, said culture medium containing a Sorangium strain
suitable for the preparation thereof, water and other suitable
customary components of culture media, and
(b) one or more cyclodextrins are added to the medium as
complex-forming component, the cyclodextrins being selected from
.alpha.-cyclodextrin, .beta.-cyclodextrin, .gamma.-cyclodextrin,
.delta.-cyclodextrin, .epsilon.-cyclodextrin, .zeta.-cyclodextrin,
.eta.-cyclodextrin and .theta.-cyclodextrin; or a cyclodextrin
derivative or a mixture of cyclodextrin derivatives selected from
derivatives of a cyclodextrin in which one or more up to all of the
hydroxy groups are etherified to an alkyl ether, an
aryl-hydroxyalkyl ether; a hydroxyalkyl ether; a carboxyalkyl
ether; a derivatized carboxy lower alkyl ether in which the
derivatized carboxy is aminocarbonyl, mono- or
di-lower-alkylaminocarbonyl, morpholino-, piperidino-, pyrrolidino-
or piperazino-carbonyl, or alkyloxycarbonyl; a sulfoalkyl ether; a
cyclodextrin in which one or more OH groups are etherified with a
radical of formula
wherein alk is alkyl and n is a whole number from and including 2
up to and including 12; a cyclodextrin in which one or more OH
groups are etherified with a radical of formula ##STR6##
wherein R' is hydrogen, hydroxy, --O--(alk--O).sub.z --H,
--O--(alk(--R)--O--).sub.p --H or --O--(alk(--R)--O--).sub.q
--alk--CO--Y; alk in all cases is alkyl; m, n, p, q and z are a
whole number from 1 to 12; and Y is OR.sub.1 or NR.sub.2 R.sub.3,
wherein R.sub.1, R.sub.2 and R.sub.3, independently of one another,
are hydrogen or lower alkyl, or R.sub.2 and R.sub.3, combined with
the binding nitrogen signify morpholino, piperidino, pyrrolidino or
piperazino; or a branched cyclodextrin in which etherifications or
acetals exist with other sugar molecules, and which are selected
from glucosyl-, diglucosyl-(G.sub.2 -.beta.-cyclodextrin),
maltosyl- and dimaltosyl-cyclodextrin, or N-acetylglucosaminyl-,
glucosaminyl-, N-acetylgalactosaminyl- and
galactosaminyl-cyclodextrin; and a lower alkanoyl-, such as
acetylester of a cyclodextrin; or mixtures of two or more of the
said cyclodextrins and/or cyclodextrin derivatives.
4. Process according to claim 3, in which the cyclodextrin and/or
the cyclodextrin derivative is added to the culture medium in a
concentration of between 0.05 and 10 percent by weight (w/v).
5. Process according to claim 4, in which the cyclodextrin and/or
the cyclodextrin derivative is added in a concentration of between
0.1 and 2 percent by weight.
6. A process according to claim 3, in which the cyclodextrin
derivative is selected from a cyclodextrin and a hydroxy lower
alkyl cyclodextrin; or mixtures of one or more thereof.
7. A process according to claim 1, in which the complex-forming
component is 2-hydroxy-propyl-.beta.-cyclodextrin.
8. A process for the production of epothilones, which process
comprises providing a culture medium for the biotechnological
preparation of these compounds, the medium comprising myxobacteria
as producers of natural substances, adding to the medium a
complex-forming component which is soluble in the culture medium,
and subsequently purifying and, if desired, separating the
epothilones from one another, wherein the complex-forming component
forms complex with the epothilones.
9. A process according to claim 8, wherein epothilone A and/or
epothilone B are obtained from a culture medium for the
biotechnological preparation of these compounds comprising a
myxobacterium of the genus Sorangium, and to which culture medium
is added a complex-forming component which is soluble in the
culture medium, comprising separating the culture into the solid
and the liquid phase (centrifugate) by centrifugation; mixing the
centrifugate with a resin or running it over a column filled with
such a resin; if necessary washing the resin with water; desorbing
the epothilone(s) from the resin with a polar solvent; if necessary
concentrating with prior, simultaneous or subsequent addition of
water; adding an organic solvent which is immiscible with water
added and transferring the epothilone(s) into the organic phase;
concentrating the organic phase obtained if required; concentrating
the epothilones from the organic solution obtained are a molecular
sieve for compounds of low molecular weight; and subsequently
making the fractions containing the epothilones undergo separation
on a reversed-phase column; whereby epothilones A and/or B are
obtained separately and, if desired, can be further concentrated by
recrystallization.
10. A process according to claim 8 for the production of epothilone
A and/or B, wherein the epothilones A and/or B are obtained from a
culture medium for the biotechnological preparation of these
compounds, to which medium is added a complex-forming component
which is soluble in the culture medium, comprising separating the
culture into the solid and the liquid phase (centrifugate) by
centrifugation; mixing the centrifugate with a resin or running it
through a column filled with such a resin; if necessary washing the
resin with water; desorbing the epothilone(s) from the resin with a
polar solvent; if necessary removing the polar solvent with prior,
simultaneous or subsequent addition of water; extracting the
resulting water phase with a solvent suitable for forming a second
phase; concentrating the organic phase obtained if required;
separating epothilone A and epothilone B from one another directly
by reversed-phase chromatography whilst eluting with an eluent
containing a nitrile; concentrating subsequently; if desired,
treating the residue from an aqueous solution by extracting once or
several times with a solvent which is immiscible with water;
dissolving it in an appropriate solvent, filtering the resulting
solution if necessary, adding the solution to a silica gel column
and eluting with an appropriate solvent or solvent mixture; and
subsequently separately combining each fraction containing
epothilone A or in particular B and concentrating it by removing
the solvent; then dissolving the residue in an appropriate alcohol,
if desired, in order to obtain especially high purity, mixing with
activated carbon and then filtering; and finally obtaining
epothilone A or B by recrystallization.
11. A process for the production of epothilones, which
comprising
a) concentrating epothilones in a culture medium for the
biotechnological preparation of these compounds, which contains a
microorganism suitable for the preparation thereof, water and other
suitable customary constituents of culture media, whereby a
cyclodextrin or a cyclodextrin derivative is added to the medium,
or a mixture of two or more of these compounds; and
b) a step for separating epothilones from one another, which is
characterised by chromatography on a reversed-phase column with an
eluant comprising a lower alkylcyanide, whereby chromatography is
carried out on column material charged with hydrocarbon chains, and
an eluant comprising a lower alkyinitrile is used; whereby if
desired further working up steps and purification steps are
possible.
12. The process of claim 11, wherein the cyclodextrin or derviative
thereof is selected from .alpha.-cyclodextrin, .beta.-cyclodextrin,
.gamma.-cyclodextrin, .delta.-cyclodextrin, .epsilon.-cyclodextrin,
.zeta.-cyclodextrin, .eta.-cyclodextrin and .theta.-cyclodextrin;
or a cyclodextrin derivative or a mixture of cyclodextrin
derivatives selected from derivatives of a cyclodextrin in which
one or more up to all of the hydroxy groups are etherified to an
alkyl ether, an aryl-hydroxyalkyl ether; a hydroxyalkyl ether; a
carboxyalkyl ether; a derivatized carboxy lower alkyl ether in
which the derivatized carboxy is aminocarbonyl, mono- or
di-lower-alkylaminocarbonyl, morpholino-, piperidino-, pyrrolidino-
or piperazino-carbonyl, or alkyloxycarbonyl; a sulfoalkyl ether; a
cyclodextrin in which one or more OH groups are etherified with a
radical of formula
wherein alk is alkyl and n is a whole number from and including 2
up to and including 12; a cyclodextrin in which one or more OH
groups are etherified with a radical of formula ##STR7##
wherein R' is hydrogen, hydroxy, --O--(alk--O).sub.z --H,
--O--(alk(--R)--O--).sub.p --H or --O--(alk(--R)--O--).sub.q
--alk--CO--Y; alk in all cases is alkyl; m, n, p, q and z are a
whole number from 1 to 12; and Y is OR.sub.1 or NR.sub.2 R.sub.3,
wherein R.sub.1, R.sub.2 and R.sub.3, independently of one another,
are hydrogen or lower alkyl, or R.sub.2 and R.sub.3, combined with
the binding nitrogen signify morpholino, piperidino, pyrrolidino or
piperazino; or a branched cyclodextrin in which etherifications or
acetals exist with other sugar molecules, and which are selected
from glucosyl-, diglucosyl-(G.sub.2 -.beta.-cyclodextrin),
maltosyl- and dimaltosyl-cyclodextrin, or N-acetylglucosaminyl-,
glucosaminyl-, N-acetylgalactosaminyl- and
galactosaminyl-cyclodextrin; and a lower alkanoyl-; or mixtures of
two or more of the cyclodextrins.
13. The process of claim 11 wherein the cyclodextrin or derivative
thereof is added to the culture medium in a concentration of
between 0.05 and 10 percent by weight (w/v).
14. The process of claim 13 wherein the cyclodextrin or derivative
thereof is added to the culture medium in a concentration of
between 0.1 and 2 percent by weight (w/v).
15. The process of claim 11 wherein the cyclodextrin derivative
comprises 2-hydroxy-propyl-.beta.-cyclodextrin.
Description
The invention relates to a new biotechnological preparation process
that can be used on an industrial scale for the production of
epothilones, especially a process for concentrating these compounds
in the culture medium, as well as a new strain for the fermentative
preparation of these compounds. The invention also relates to new
crystal forms of epothilones, especially epothilone B, obtainable
by the new processes, their usage in the production of
pharmaceutical preparations, new pharmaceutical preparations
comprising these new crystal forms and/or the use of these
compounds in the treatment of proliferative diseases such as
tumours, or in the production of pharmaceutical formulations which
are suitable for this treatment.
BACKGROUND OF THE INVENTION
Of the existing cytotoxic active ingredients for treating tumours,
Taxol.RTM. (Paclitaxel; Bristol-Myers Squibb), a
microtubuli-stabilising agent, plays an important role and has
remarkable commercial success. However, Taxol has a number of
disadvantages. In particular, its very poor solubility in water is
a problem. It therefore became necessary to administer Taxol.RTM.
in a formulation with Cremophor EL.RTM. (polyoxyethylated castor
oil; BASF, Ludwigshafen, Germany). Cremophor EL.RTM. has severe
side effects; for example it causes allergies which in at least one
case have led even to the death of a patient.
Although the Taxan class of microtubuli-stabilising anti-cancer
agents has been commended as "perhaps the most important addition
to the pharmaceutical armoury against cancer in the last decade"
(see Rowinsky E. K., Ann. rev. Med. 48, 353-374 (1997)), and
despite the commercial success of Taxol.RTM., these compounds still
do not appear to represent a really great breakthrough in the
chemotherapy of cancer. Treatment with Taxol.RTM. is linked with a
series of significant side effects, and a few primary classes of
compact tumours, namely colon and prostate tumours, respond to this
compound only to a small extent (see Rowinsky E. K., inter alia).
In addition, the efficacy of Taxol can be impaired and even
completely neutralised by acquired resistance mechanisms,
especially those based on the overexpression of phosphoproteins,
which act as efflux pumps for active ingredients, such as
"Multidrug Resistance" due to overexpression of the multidrug
transport glycoprotein "P-glycoprotein".
Epothilones A and B represent a new class of
microtubuli-stabilising cytotoxic active ingredients (see Gerth, K.
et al., J. Antibiot. 49, 560-3 (1966)) of the formulae:
##STR1##
wherein R signifies hydrogen (epothilone A) or methyl (epothilone
B).
These compounds have the following advantages over Taxol.RTM.:
a) They have better water-solubility and are thus more easily
accessible for formulations.
b) It has been reported that, in cell culture experiments, they are
also active against the proliferation of cells, which, owing to the
activity of the P-glycoprotein efflux pump making them "multidrug
resistant", show resistance to treatment with other chemotherapy
agents including Taxol.RTM. (see Bolag, D. M., et al.,
"Epothilones, a new class of microtubuli-stabilizing agents with a
Taxol-like mechanism of action", Cancer Research 55, 2325-33
(1995)). And
c) it could be shown that they are still very effective in vitro
against a Taxol.RTM.-resistant ovarian carcinoma cell line with
modified .beta.-tubulin (see Kowalski, R. J., et al., J. Biol.
Chem. 272(4), 2534-2541 (1997)).
Pharmaceutical application of the epothilones, for example for
tumour treatment, is possible in an analogous manner to that
described for Taxol, see for example U.S. Pat. No. 5,641,803; U.S.
Pat. No. 5,496,804; U.S. Pat. No. 5,565,478).
In order to be able to use the epothilones on a larger scale for
pharmaceutical purposes, however, it is necessary to obtain
appropriate amounts of these compounds.
Until now, the extraction of natural substances by means of
myxobacteria, especially the epothilones from the cell strain
Sorangium cellulosum Soce90 (deposited under no. 6773 at the German
Collection of Microorganisms, see WO 93/10121) was described in
literature. In order to obtain a satisfactory concentration of the
natural substances, especially the epothilones, in the culture
medium for the subsequent extraction, previously an adsorbate resin
based on polystyrene was always added, for example Amberlite
XAD-1180 (Rohm & Haas, Frankfurt, Germany).
However, the disadvantage of this process is that, on a large
scale, it leads to an abundance of problems. Valves are impaired by
the globules of resin, pipes can block, and apparatus may be
subject to greater wear due to mechanical friction. The globules of
resin are porous and therefore have a large inner surface area
(about 825 m.sup.2 /gram resin). Sterilisation becomes a problem,
as air enclosed in the resin is not autoclaved. Thus, the process
cannot be practicably carried out on a large scale using resin
addition.
On the other hand, without adding resin globules, a satisfactory
concentration of epothilones cannot be achieved in the culture
medium.
Surprisingly, the requirements for finding a way out of this
dilemma have now been found, enabling a satisfactory concentration
of natural substances to be obtained from microorganisms, in
particular myxobacteria, which produce epothilones such as
epothilone A or B, in particular a concentration of epothilones A
and B, in the culture medium, without the addition of resins, and
thus enabling production of these compounds, especially epothilones
to be carried out on a technical and industrial scale without the
above-mentioned disadvantages.
DETAILED DESCRIPTION OF THE INVENTION
One aspect of the invention relates to a process for concentrating
epothilones, especially epothilone A and/or B, in particular
epothilone B, in a culture medium, in order to produce these
compounds on a biotechnological scale, the process comprising
microorganisms which produce these compounds, especially
myxobacteria (as producers of natural substances), whereby a
complex-forming components which is soluble in the culture medium
is added to the medium.
A further aspect relates to the corresponding culture medium, which
comprises a corresponding complex-forming component and
microorganisms, especially myxobacteria, in particular of the genus
Sorangium, which are suitable for producing epothilones, especially
epothilone A and/or B.
A further aspect of the invention relates to a process for the
production of epothilones, especially epothilone A and/or B,
especially the two pure compounds, in particular epothilone B,
which is characterised in that the epothilones are obtained by
working up a culture medium for the biotechnological preparation of
these compounds, which comprises as producers of natural substances
microorganisms, especially myxobacteria, that produce these
compounds, and to which a complex-forming component that is soluble
in the culture medium is added, and the subsequent purification
and, if desired, separation of the epothilones, for example
epothilone A and B.
A fourth aspect of the invention relates to a method of separating
epothilones, especially epothilones A and B from one another, which
is characterised by chromatography on a reversed-phase column with
an eluant comprising a lower alkyl cyanide.
A further aspect of the invention relates to a strain of Sorangium
cellulosum obtained by mutagenesis, which under otherwise identical
conditions, produces more epothilones than Sorangium cellulosum
Soce90.
A further aspect also relates to new crystal forms of epothilone
B.
The general terms used hereinabove and hereinbelow preferably have
the meanings given hereinbelow:
Where reference is made hereinabove and hereinbelow to documents,
these are incorporated insofar as is necessary.
The prefix "lower" always indicates that the correspondingly named
radical contains preferably up to a maximum of 7 carbon atoms, in
particular up to 4 carbon atoms, and is branched or unbranched.
Lower alkyl may be for example unbranched or branched once or more,
and is e.g. methyl, ethyl, propyl such as isopropyl or n-propyl,
butyl such as isobutyl, sec.-butyl, tert.-butyl or n-butyl, or also
pentyl such as amyl or n-pentyl.
A culture medium for the biotechnological preparation of
epothilones which contains microorganisms that produce these
compounds, especially myxobacteria, as producers of natural
substances, is primarily a medium which comprises a corresponding
(naturally occurring or also obtainable by cultivation or in
particular by genetic modification) microorganism, especially a
myxobacterial strain which is in a position to produce these
compounds, in particular a myxobacterium of the genus Sorangium,
preferably (in particular for epothilone production) a
microorganism of the type Sorangium cellulosum Soce90 (see WO
93/10121), or a biomaterial derived therefrom or from parts of this
myxobacterium, especially a correspondingly derived strain, in
particular the strain having the reference BCE33/10, in particular
the strain having the reference BCE 63/114 or mutants thereof, and
in addition, together with water, preferably other conventional and
appropriate constituents of culture media, such as biopolymers,
sugar, amino acids, salts, nucleic acids, vitamins, antibiotics,
semiochemicals, growth media, extracts from biomaterials such as
yeast or other cell extracts, soy meal, starch such as potato
starch and/or trace elements, for example iron ions in
complex-bound form, or suitable combinations of all or some of
these constituents and/or also analogous additions. The
corresponding culture media are known to the person skilled in the
art or may be produced by known processes (see e.g. the culture
media in the examples of the present disclosure, or in WO
93/10121).
One preferred myxobacterium is a strain selected by UV mutagenesis
and selection for increased formation of epothilone A and/or B over
Sorangium cellulosum Soce90, which is deposited in the DSM under
no. 6773, especially the mutant BCE33/10, which was deposited under
the number DSM 11999 on Feb. 9, 1998 at the German Collection of
Microorganisms and Cell Cultures (DSMZ Mascheroder Weg 1b, D-38124,
Braunschweig, Germany), and most preferably the mutant having the
reference BCE 63/114, which was deposited under number DSM 12539 on
Nov. 27, 1998 at the German Collection of Microorganisms and Cell
Cultures (DSMZ).
Strain culture and morphological description of strain BCE 33/10
and of strain BCE 63/114: The strain can grow on cellulose as the
sole source of carbon and energy with potassium nitrate as the sole
source of nitrogen, e.g. on filter paper over ST21 mineral salt
agar (0.1% KNO.sub.3 ; 0.1% MgSO.sub.4.times.7 H.sub.2 O; 0.1%
CaCl.sub.2.times.2 H.sub.2 O; 0.1% K.sub.2 HPO.sub.4 ; 0.01%
MnSO.sub.4.times.7 H.sub.2 O; 0.02% FeCl.sub.3 ; 0.002% yeast
extract; standard trace element solution; 1% agar). On this medium,
dark reddish-brown to blackish-brown fruiting bodies are formed.
They consist of small sporangioles (ca. 15 to 30 .mu.m diameter)
and exist in dense heaps and packs of varying size.
The vegetative bacilli have the shape typical of Sorangium
(relatively compact, under the phase contrast microscope dark,
cylindrical bacilli with broad rounded ends, on average 3 to 6
.mu.m long and 1 .mu.m thick).
Epothilones are primarily epothilone A and/or B, but also other
epothilones, for example epothilones C and D named in International
Application WO 97/19086 and WO 98/22461, epothilones E and F named
in WO 98/22461, and further epothilones obtainable from
corresponding microorganisms.
A water-soluble complex-forming component is primarily a
water-soluble oligo- or poly-peptide derivative or in particular an
oligo- or polysaccharide derivative of cyclic or helical structure,
which forms an intramolecular cavity, which because of its
sufficiently hydrophobic properties is in a position to bind
epothilones, especially epothilone A and/or epothilone B. A
water-soluble complex-forming component that is especially
preferred is one that is selected from cyclodextrins or (in
particular) cyclodextrin derivatives, or mixtures thereof.
Cyclodextrins are cyclic (.alpha.-1,4)-linked oligosaccharides of
.alpha.-D-glucopyranose with a relatively hydrophobic central
cavity and a hydrophilic external surface area.
The following are distinguished in particular (the figures in
parenthesis give the number of glucose units per molecule):
.alpha.-cyclodextrin (6), .beta.-cyclodextrin (7), .gamma.-
cyclodextrin (8), .delta.-cyclodextrin (9), .epsilon.-cyclodextrin
(10), .zeta.-cyclodextrin (11), .eta.-cyclodextrin (12), and
.theta.-cyclodextrin (13). Especially preferred are
.delta.-cyclodextrin and in particular .alpha.-cyclodextrin,
.beta.-cyclodextrin or .gamma.-cyclodextrin, or mixtures
thereof.
Cyclodextrin derivatives are primarily derivatives of the
above-mentioned cyclodextrins, especially of .alpha.-cyclodextrin,
.beta.-cyclodextrin or .gamma.-cyclodextrin, primarily those in
which one or more up to all of the hydroxy groups (3 per glucose
radical) are etherified or esterified. Ethers are primarily alkyl
ethers, especially lower alkyl, such as methyl or ethyl ether, also
propyl or butyl ether; the aryl-hydroxyalkyl ethers, such as
phenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether;
the hydroxyalkyl ethers, in particular hydroxy-lower-alkyl ethers,
especially 2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or
hydroxy-butyl such as 2-hydroxybutyl ether; the carboxyalkyl
ethers, in particular carboxy-lower-alkyl ethers, especially
carboxymethyl or carboxyethyl ether; derivatised carboxyalkyl
ethers, in particular derivatised carboxy-lower-alkyl ether in
which the derivatised carboxy is etherified or amidated carboxy
(primarily aminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl,
morpholino-, piperidino-, pyrrolidino- or piperazino-carbonyl, or
alkyloxycarbonyl), in particular lower alkoxycarbonyl-lower-alkyl
ether, for example methyloxycarbonylpropyl ether or
ethyloxycarbonylpropyl ether; the sulfoalkyl ethers, in particular
sulfo-lower-alkyl ethers, especially sulfobutyl ether;
cyclodextrins in which one or more OH groups are etherified with a
radical of formula
wherein alk is alkyl, especially lower alkyl, and n is a whole
number from 2 to 12, especially 2 to 5, in particular 2 or 3;
cyclodextrins in which one or more OH groups are etherified with a
radical of formula ##STR2##
wherein R' is hydrogen, hydroxy, --O--(alk--O).sub.z --H,
--O--(alk(--R)--O--).sub.p --H or --O--(alk(--R)--O--).sub.q
--alk--CO--Y; alk in all cases is alkyl, especially lower alkyl; m,
n, p, q and z are a whole number from 1 to 12, preferably 1 to 5,
in particular 1 to 3; and Y is OR.sub.1 or NR.sub.2 R.sub.3,
wherein R.sub.1, R.sub.2 and R.sub.3 independently of one another,
are hydrogen or lower alkyl, or R.sub.2 and R.sub.3 combined
together with the linking nitrogen signify morpholino, piperidino,
pyrrolidino or piperazine;
or branched cyclodextrins, in which etherifications or acetals with
other sugars are present, especially glucosyl-, diglucosyl-(G.sub.2
-.beta.-cyclodextrin), maltosyl- or dimaltosyl-cyclodextrin, or
N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- or
galactosaminyl- cyclodextrin.
Esters are primarily alkanoyl esters, in particular lower alkanoyl
esters, such as acetyl esters of cyclodextrins.
It is also possible to have cyclodextrins in which two or more
different said ether and ester groups are present at the same
time.
Mixtures of two or more of the said cyclodextrins and/or
cyclodextrin derivatives may also exist.
Preference is given in particular to .alpha.-, .beta.- or
.gamma.-cyclodextrins or the lower alkyl ethers thereof, such as
methyl-.beta.-cyclodextrin or in particular
2,6-di-O-methyl-.beta.-cyclodextrin, or in particular the hydroxy
lower alkyl ethers thereof, such as 2-hydroxypropyl-.alpha.-,
2-hydroxypropyl-.beta.- or
2-hydroxypropyl-.gamma.-cyclodextrin.
The cyclodextrins or cyclodextrin derivatives are added to the
culture medium preferably in a concentration of 0.02 to 10,
preferably 0.05 to 5, especially 0.1 to 4, for example 0.1 to 2
percent by weight (w/v).
Cyclodextrins or cyclodextrin derivatives are known or may be
produced by known processes (see for example U.S. Pat. No.
3,459,731; U.S. Pat. No. 4,383,992; U.S. Pat. No. 4,535,152; U.S.
Pat. No. 4,659,696; EP 0 094 157; EP 0 149 197; EP 0 197 571; EP 0
300 526; EP 0 320 032; EP 0 499 322; EP 0 503 710; EP 0 818 469; WO
90/12035; WO 91/11200; WO 93/19061; WO 95/08993; WO 96/14090; GB
2,189,245; DE 3,118,218; DE 3,317,064 and the references mentioned
therein, which also refer to the synthesis of cyclodextrins or
cyclodextrin derivatives, or also: T. Loftsson and M. E. Brewster
(1996): Pharmaceutical Applications of Cyclodextrins: Drug
Solubilization and Stabilisation: Journal of Pharmaceutical Science
85 (10):1017-1025; R. A. Rajewski and V. J. Stella(1996):
Pharmaceutical Applications of Cyclodextrins: In Vivo Drug
Delivery: Journal of Pharmaceutical Science 85 (11):
1142-1169).
In the following description of the working up, "epothilone" is
understood to be an epothilone which is obtainable from the
corresponding microorganism, preferably epothilone C, D, E, F or
especially A or in particular epothilone B. If not otherwise
stated, where "epothilones" are mentioned, this is intended to be a
general term which includes individual epothilones or mixtures.
Working up of the epothilones is effected by conventional methods;
first of all, by separating a culture into the liquid phase
(centrifugate or filtrate) and solid phase (cells) by means of
filtration or centrifugation (tubular centrifuge or separator). The
(main) part of the epothilones found in the centrifugate or in the
filtrate is then directly mixed with a synthetic resin, for example
a resin based on polystyrene as matrix (hereinafter referred to
also simply as polystyrene resin), such as Amberlite XAD-16 [Rohm
& Haas Germany GmbH, Frankfurt] or Diaion HP-20 [Resindion
S.R.L., Mitsubishi Chemical Co., Milan] (preferably in a ratio of
centrifugate: resin volume of ca. 10:1 to 100:1, preferably about
50:1). After a period of contact of preferably 0.25 to 50 hours,
especially 0.8 to 22 hours, the resin is separated, for example by
filtration or centrifugation. If required, after adsorption, the
resin is washed with a strongly polar solvent, preferably with
water. Desorption of the epothilones is then effected with an
appropriate solvent, especially with an alcohol, in particular
isopropanol. The solvent phase, especially the isopropanol phase,
is then removed from the solvent, preferably by means of prior,
simultaneous or subsequent addition of water, in particular in a
circulating evaporator, thereby being concentrated if necessary,
and the resulting water phase is extracted with a solvent suitable
for forming a second phase, such as an ester, for example a lower
alkanol lower alkanoate, typically ethyl acetate or isopropyl
acetate. The epothilones are thereby transferred into the organic
phase. Then the organic phase is concentrated to the extent
necessary, preferably to dryness, for example using a rotary
evaporator.
Subsequently, further processing takes place using the following
steps, whereby the purification step by means of reversed-phase
chromatography with elution with a nitrile is an inventive step and
is thus compulsory, while the other steps are optional:
molecular filtration (gel chromatography), e.g. on a column of
material such as Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with
an alcohol such as methanol as eluant;
separation of the epothilones by reversed-phase chromatography
after being taken up in a suitable solvent, and elution with a
mixture of nitrile/water (compulsory), preferably characterised in
that the chromatography is carried out on a column of material,
especially a RP-18 material, which is charged with hydrocarbon
chains, such as hydrocarbon chains containing 18 carbon atoms, and
an eluant comprising a nitrile, especially a lower alkyl-nitrile,
in particular acetonitrile, is used, in particular a mixture of
nitrile/water is used, especially a mixture of acetonitrile/water,
preferably in a ratio of nitrile to water of about 1:99 to 99:1,
primarily between 1:9 and 9:1, e.g. between 2:8 and 7:3, e.g. 3:7
or 4:6.
single or multiple extraction of the residue (especially after
evaporation) in a two-phase system consisting of water and a
solvent immiscible with water, preferably an ester, in particular a
lower alkyl lower alkanoate, such as ethyl acetate or isopropyl
acetate;
adsorption chromatography, in particular by adding to a column of
silica gel and eluting with an appropriate solvent or solvent
mixture, especially a mixture of ester/hydrocarbon, for example
lower alkyl alkanoate/C.sub.4 -C.sub.10 -alkane, especially ethyl
or isopropyl acetate/n-hexane, in which the ratio between the ester
and hydrocarbon is preferably in the range 99:1 to 1:99, preferably
10:1 to 1:10, for example 4:1;
dissolving the residue, which may be obtained after concentration,
in an appropriate solvent such as an alcohol, e.g. methanol;
mixing with activated carbon and removal thereof;
recrystallisation, e.g. from appropriate solvents or solvent
mixtures, for example consisting of esters, ester/hydrocarbon
mixtures or alcohols, especially ethyl or isopropyl acetate:
toluene 1:10 to 10:1, preferably 2:3 (epothilone A) or methanol or
ethyl acetate (epothilone B);
whereby between each step being employed, the resulting solutions
or suspensions are concentrated if necessary, and/or liquid and
solid components are separated from one another, in particular by
filtering or centrifuging solutions/suspensions. The more precise
definitions mentioned below can be preferably used in the above
individual steps.
The further working up and purification is preferably carried
out
either by direct separation of the epothilones from one another by
reversed-phase chromatography after being taken up in an
appropriate solvent, for example a nitrile/water mixture,
especially an acetonitrile/water mixture (ratio of nitrile to water
1:99 to 99:1, preferably 1:9 to 9:1, especially 3:1), if necessary
after filtration or centrifugation, preferably on a silica gel that
has been derivatized by hydrocarbon radicals, e.g. a silica gel
modified by alkyl radicals containing 8 to 20, especially 18,
C-atoms, eluting with an eluant comprising a nitrile, especially a
lower alkylnitrile, such as acetonitrile, especially a mixture of
the nitrile with water, such as an acetonitrile/water mixture,
whereby detection of the interesting fractions is effected in
conventional manner, for example by UV detection or (preferably) by
on-line HPLC (HPLC with a very small column, the analyses taking
less than 1 minute, and detection e.g. at 250 nm), this enabling a
particularly exact separation of the fractions containing the
desired product to take place; if required, with subsequent
concentration, for example by distillation, to remove the nitrile;
if desired, with subsequent single or multiple, for example double,
extraction of the residue of evaporation in a two-phase system
consisting of water and an immiscible solvent, such as ethyl
acetate or isopropyl acetate; additional concentration of the
organic phase and dissolving of the residue in an appropriate
solvent, preferably an ester such as ethyl acetate or isopropyl
acetate, if required, filtration or centrifugation, if desired
adding to a column of silica gel and eluting with an appropriate
solvent or solvent mixture, for example with a mixture of
ester/hydrocarbon, e.g. lower alkyl alkanoate/C.sub.4 -C.sub.10
-alkane, especially ethyl or isopropyl acetate/n-hexane, in which
the ratio of ester to hydrocarbon is preferably in the range 99:1
to 1:99, preferably 10:1 to 1:10, e.g. 4:1; subsequent combining of
the fractions containing each desired epothilone, especially
epothilone A or epothilone B, and after removing the solvent, for
example by distillation, preferably concentrating to dryness; then,
dissolving of the residue in an appropriate alcohol, preferably
methanol; and if desired, in order to obtain especially high
purity, mixing with activated carbon and then separating the
activated carbon, for example by filtration; and finally, by
recrystallisation as described below under variant 2 (for
epothilone B in particular from methanol), separate extraction of
the epothilones, especially epothilones A or B. This is the most
preferred variant 1, the outstanding characteristic of which is the
surprising direct separation by reversed-phase chromatography of
the epothilone-containing mixture desorbed by the resin, despite
all the impurities in the organic extract;
or (variant 2) first of all exclusion chromatography takes place
(molecular filtration) e.g. on a column of material such as
Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with an alcohol such as
methanol as eluant, and then subsequent separation of the
epothilones present in the peak fractions obtained, e.g. epothilone
A and B, by reversed-phase chromatography as described above for
variant 1; if required twice, if peak fractions of one epothilone
contain those of another, for example if those with epothilone A
still contain residues of epothilone B; and then separate
recrystallisation of each epothilone from appropriate solvents or
solvent mixtures, for example from ethyl or isopropyl
acetate:toluene 1:10 to 10:1, preferably 2:3 (epothilone A) or
methanol or ethyl acetate (epothilone B). This is variant 2 of
working up and purification.
With variant 1, highly pure epothilone B may be obtained in a
relatively simpler manner (the purity is preferably greater than
97%, especially over 99%).
Variant 1 preferably takes place as follows (whereby preferably the
above-mentioned variants can be used instead of the following
general definitions): First of all, a culture is separated into the
liquid phase (centrifugate or filtrate) and a solid phase (cells)
by means of filtration or centrifugation (tubular centrifuge or
separator). The (main) part of the epothilones found in the
centrifugate or filtrate is then directly mixed with a synthetic
resin. After a contact period of preferably 0.25 to 50 hours, the
resin is separated, for example by filtration or centrifugation. If
required, after adsorption, the resin is washed with a strongly
polar solvent, preferably with water. Desorption of the epothilones
is then effected with an appropriate solvent, especially with an
alcohol, in particular isopropanol. The solvent phase, especially
isopropanol phase, is then removed from the solvent, preferably by
means of prior, simultaneous or subsequent addition of water, in
particular in a circulating evaporator, thereby being concentrated
if necessary, and the resulting water phase is extracted with a
solvent suitable for forming a second phase, such as an ester, for
example a lower alkanol lower alkanoate, typically ethyl acetate or
isopropyl acetate. The epothilones are then transferred into the
organic phase. Then the organic phase is concentrated to the extent
necessary, preferably to dryness, for example using a rotary
evaporator. By subsequent reversed-phase chromatography on a silica
gel derivatized with hydrocarbon atoms, e.g. a silica gel modified
by alkyl radicals containing 18 C-atoms, and eluting with a mixture
of a lower alkylnitrile such as acetonitrile with water, the
epothilones are directly separated from one another, especially
epothilone A and epothilone B; then, concentration takes place by
means of distillation, the residue is shaken out once or more, if
desired from water with an appropriate solvent that is immiscible
with water, preferably an ester such as isopropyl acetate, then the
organic phase is again concentrated and the residue of evaporation
is dissolved in an ester such as ethyl or isopropyl acetate,
filtered if required, the filtrate added to a column of silica gel,
and eluted with a mixture of ester/hydrocarbon, e.g. ethyl or
isopropyl acetate/n-hexane; subsequently, the fractions containing
the epothilone, especially epothilone A or B, are respectively
combined and, after removing the solvent by distillation,
concentrated, preferably to dryness; the residue is then dissolved
in an appropriate lower alkanol, preferably methanol, and in order
to obtain especially high purity, mixed with activated carbon and
then filtered; finally, the epothilones are extracted by
recrystallisation (in the case of epothilone B preferably from
methanol).
Variant 2 is effected preferably as follows: After harvest, a
culture is separated into the liquid phase (centrifugate) and solid
phase (cells) by means of centrifugation (tubular centrifuge or
separator). The main part of the epothilones are found in the
centrifugate, which is then directly mixed with a polystyrene
resin, such as Amberlite XAD-16 [Rohm & Haas Germany GmbH,
Frankfurt] or Diaion HP-20 [Resindion S.R.L., Mitsubishi Chemical
Co., Milan] (preferably in a ratio of centrifugate: resin volume of
ca. 10:1 to 100:1, preferably about 50:1) and stirred in an
agitator. In this step, the epothilones are transferred from the
cyclodextrin to the resin. After a period of contact of ca. 1 hour,
the resin is separated by centrifugation or filtration. Adsorption
of the epothilones onto the resin may also be effected in a
chromatography column, by placing the resin in the column and
running the centrifugate over the resin. After adsorption, the
resin is washed with water. Desorption of the epothilones from the
resin is effected with isopropanol. The isopropanol phase is then
freed of isopropanol preferably by the addition of water in
particular in a circulating evaporator, and the resulting water
phase is extracted with ethyl acetate. The epothilones are thus
transferred from the water phase to the ethyl acetate phase. Then
the ethyl acetate extract is concentrated to dryness, for example
using a rotary evaporator. An initial concentration of the
epothilones is then achieved by means of molecular filtration (e.g.
Sephadex LH-20 [Pharmacia, Uppsala, Sweden] with methanol as
eluant). The peak fractions from the molecular filtration contain
epothilone A and B and have a total epothilone content of >10%.
Separation of these peak fractions, which contain epothilone A and
B in a mixture, into the individual components, then follows by
means of chromatography on a "reversed-phase", e.g. RP-18 phase
(phase which is modified by alkyl radicals containing 18 carbon
atoms per chain), with an appropriate eluant, preferably one
containing a nitrile such as acetonitrile (this gives better
separation than for example alcohols such as methanol). Epothilone
A elutes before epothilone B. The peak fractions with epothilone B
may still contain small portions of epothilone A, which can be
removed by further separation on RP-18. Finally, the epothilone A
fraction is crystallised directly from ethyl acetate:toluene=2:3,
and the epothilone B fraction from methanol or ethyl acetate.
Preferred Embodiment of the Invention
The invention preferably relates to a process for the concentration
of epothilones, especially epothilone A and/or B, in particular
epothilone B, in a culture medium for the biotechnological
preparation of this (these) compound(s), which contains a
microorganism which is suitable for this preparation, especially a
Sorangium strain, especially of the type Sorangium cellulosum
Soce90, or a mutant arising therefrom, in particular the strain
having reference BCE 33/10, especially the strain having reference
BCE 63/114, water and other usual appropriate constituents of
culture media, whereby a cyclodextrin or a cyclodextrin derivative,
or a mixture of two or more of these compounds is added to the
medium, especially one or more, preferably one or two or more
cyclodextrins selected from .alpha.-cyclodextrin (6),
.beta.-cyclodextrin (7), .gamma.-cyclodextrin (8),
.delta.-cyclodextrin (9), .epsilon.-cyclodextrin (10),
.zeta.-cyclodextrin (11), .eta.-cyclodextrin (12), and
.theta.-cyclodextrin (13), especially .alpha.-cyclodextrin,
.beta.-cyclodextrin or .gamma.-cyclodextrin; or primarily a
cyclodextrin derivative or mixture of cyclodextrin derivatives
selected from derivatives of a cyclodextrin, in which one or more
up to all of the hydroxy groups are etherified to an alkyl ether,
especially lower alkyl, such as methyl or ethyl ether, also propyl
or butyl ether; an aryl-hydroxyalkyl ether, such as
phenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; a
hydroxyalkyl ether, in particular hydroxy-lower-alkyl ethers,
especially 2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or
hydroxybutyl such as 2-hydroxybutyl ether; a carboxyalkyl ether, in
particular carboxy-lower-alkyl ether, especially carboxymethyl or
carboxyethyl ether; a derivatised carboxyalkyl ether, in particular
a derivatised carboxy-lower-alkyl ether in which the derivatised
carboxy is aminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl,
morpholino-, piperidino-, pyrrolidino- or piperazino-carbonyl, or
alkyloxycarbonyl, in particular lower alkoxycarbonyl, such as
preferably lower alkoxycarbonyl-lower-alkyl ether, for example
methyloxycarbonylpropyl ether or ethyloxycarbonylpropyl ether; a
sulfoalkyl ether, in particular sulfo-lower-alkyl ether, especially
sulfobutyl ether; a cyclodextrin in which one or more OH groups are
etherified with a radical of formula
wherein alk is alkyl, especially lower alkyl, and n is a whole
number from 2 to 12, especially 2 to 5, in particular 2 or 3; a
cyclodextrin in which one or more OH groups are etherified with a
radical of formula ##STR3##
wherein R' is hydrogen, hydroxy, --O--(alk--O).sub.z --H,
--O--(Alk(--R)--O--).sub.p --H or --O--(alk(--R)--O--).sub.q
--alk--CO--Y; alk in all cases is alkyl, especially lower alkyl; m,
n, p, q and z are a whole number from 1 to 12, preferably 1 to 5,
in particular 1 to 3; and Y is OR.sub.1 or NR.sub.2 R.sub.3,
wherein R.sub.1, R.sub.2 and R.sub.3 independently of one another,
are hydrogen or lower alkyl, or R.sub.2 and R.sub.3 combined
together with the linking nitrogen signify morpholino, piperidino,
pyrrolidino or piperazino; or a branched cyclodextrin, in which
etherifications or acetals with other sugars are present, and which
are selected from glucosyl-, diglucosyl-(G.sub.2
-.beta.-cyclodextrin), maltosyl- or dimaltosyl-cyclodextrin, or
N-acetylglucosaminyl-, glucosaminyl-, N-acetylgalactosaminyl- and
galactosaminyl-cyclodextrin; or a lower alkanoyl, such as acetyl
ester of a cyclodextrin.
Particular preference is given to a process in which the
cyclodextrin and/or the cyclodextrin derivative is added to the
culture medium in a concentration of 0.02 to 10, preferably 0.005
to 10, more preferably 0.05 to 5, most preferably 0.1 to 4, for
example 0.1 to 2, percent by weight (w/v).
Especially preferred is a process according to one of the two
previous paragraphs, in which the cyclodextrin derivative is
selected from a cyclodextrin, especially .beta.-cyclodextrin, and a
hydroxy lower alkyl-cyclodextrin, especially
2-hydroxypropyl-.alpha.-, -.beta.- or -.gamma.-cyclodextrin; or
mixtures of one or more thereof; whereby
2-hydroxypropyl-.beta.-cyclodextrin on its own is preferred in
particular.
The invention also relates in particular to a culture medium, which
comprises a cyclodextrin, a cyclodextrin derivative or a mixture of
two or more complex-forming components selected from cyclodextrins
and cyclodextrin derivatives, especially a cyclodextrin or
cyclodextrin derivative as defined in the third-last paragraph, in
particular as in the second-last paragraph, or a mixture of one or
more of these compounds, and a microorganism which is suitable for
producing epothilones, especially epothilone A and/or B, preferably
a strain from the genus Sorangium, especially a strain of Sorangium
cellulosum, e.g. the strain Soce90 or a mutant arising therefrom,
in particular the strain BCE 33/10, or especially BCE 63/114.
A further aspect of the invention relates to a process for the
production of epothilone A and/or B, especially the two pure
compounds, in particular epothilone B, which is characterised in
that the epothilones are separated for example by centrifugation
into the solid and the liquid phase (centrifugate) by working up a
culture medium for the biotechnological preparation of these
compounds, as described above, to which has been added a
complex-forming component which is soluble in the culture medium,
in particular a cyclodextrin, a cyclodextrin derivative or a
mixture of two or more cyclodextrins and/or cyclodextrin
derivatives; the centrifugate is mixed with a resin, especially a
polystyrene resin, or is run through a column filled with such a
resin; if necessary, the resin is washed with water; the
epothilone(s) is or are desorbed from the resin using a polar
solvent, especially an alcohol, primarily a lower alkanol such as
isopropanol; if necessary, concentrated by means of prior,
simultaneous or subsequent addition of water; an organic solvent
which is immiscible with water, for example an ester, such as ethyl
acetate, is added, and the epothilone(s) is or are transferred to
the organic phase, for example by agitating or stirring; where
necessary, the organic phase is concentrated; the epothilones from
the organic solution obtained are concentrated through a molecular
sieve for compounds of low molecular weight; and then the fractions
containing the epothilones, especially epothilone A and/or B
undergo separation by a reversed-phase column, preferably eluting
with an eluant containing a nitrile, such as acetonitrile (or
instead, an eluant containing an alcohol, such as methanol);
whereby epothilones A and B are extracted separately, and if
desired, can be further concentrated by recrystallisation.
One preferred aspect of the invention relates to a process for the
production of epothilone A and/or B, especially the two pure
compounds, in particular epothilone B, which is characterised in
that the epothilones are separated for example by centrifugation
into the solid and the liquid phase (centrifugate) by working up a
culture medium for the biotechnological preparation of these
compounds, as described above, to which has been added a
complex-forming component which is soluble in the culture medium,
in particular a cyclodextrin, a cyclodextrin derivative or a
mixture of two or more cyclodextrins and/or cyclodextrin
derivatives; the centrifugate is mixed with a resin, especially a
polystyrene resin, or is run through a column filled with such a
resin; if necessary, the resin is washed with water; the
epothilone(s) is or are desorbed from the resin using a polar
solvent, especially an alcohol, primarily a lower alkanol such as
isopropanol; if necessary, the polar solvent is removed by means of
prior, simultaneous or subsequent addition of water; the resulting
water phase is extracted with a solvent which is suitable for
forming a second phase, for example an ester, such as diethyl
ester, if necessary, the organic phase is concentrated, preferably
to dryness; epothilone A and B are separated from one another
directly by reversed-phase chromatography, eluting with an eluant
containing a nitrile, especially a lower alkylnitrile, such as
acetonitrile, whereby detection is effected in the usual manner,
for example by UV detection or preferably by on-line HPLC (HPLC
with a very small column, the analyses taking less than 1 minute,
and detection e.g. at 250 nm); subsequent concentration, for
example by distillation; if desired, the residue is treated from an
aqueous solution once or more (for example twice) by extraction
with a solvent which is immiscible with water, such as an ester;
dissolved in an appropriate solvent, preferably an ester such as
ethyl or isopropyl acetate, filtered if necessary, added to a
column of silica gel and eluted with an appropriate solvent or
solvent mixture, for example with an ester/hydrocarbon mixture; and
subsequently, the fractions containing either epothilone A or
especially B are separately combined and, after removing the
solvent, for example by distillation, concentrated preferably to
dryness; then the residue is dissolved in an appropriate alcohol,
preferably methanol, then if desired, in order to obtain especially
high purity, treated with activated carbon and then filtered; and
finally epothilone A or B is obtained by recrystallisation (in the
case of epothilone B, particularly from methanol).
A further preferred aspect of the invention relates to a method of
separating epothilones, especially epothilones A and B from one
another, which is characterised by chromatography on a
reversed-phase column with an eluant containing a lower alkyl
cyanide, chromatography being carried out on a column material,
especially an RP-18 material, which is charged with hydrocarbon
chains containing 18 carbon atoms, and employing an eluant
containing a nitrile, especially a lower alkylnitrile, in
particular acetonitrile, especially a mixture of nitrile/water, in
particular a mixture of acetonitrile/water, preferably in a ratio
of nitrile to water of ca. 1:99 to 99:1, primarily 1:9 to 9:1, e.g.
between 2:8 and 7:3, e.g. 3:7 or 4:6. This separation may follow on
to a filtration with a molecular sieve, or is preferably effected
directly using the residue after adsorption of the epothilones from
the culture medium containing a complex-forming component onto a
resin, as described above ("variant 1") One advantage of separation
with an eluant containing a lower alkylcyanide over that using
alcohols, such as methanol, is the better separation of epothilones
A and B.
The invention relates preferably to a process for the preparation
of epothilones, which a) comprises a process for the concentration
of epothilones, especially epothilone A and/or B, in particular
epothilone B, in a culture medium for the biotechnological
preparation of this (these) compound(s), which contains a
microorganism which is suitable for this preparation, especially a
Sorangium strain, especially of the type Sorangium cellulosum
Soce90, or a mutant arising therefrom, in particular the strain
having reference BCE 33/10, especially the strain having reference
BCE 63/114, water and other usual appropriate constituents of
culture media, whereby a cyclodextrin or a cyclodextrin derivative,
or a mixture of two or more of these compounds is added to the
medium, especially one or more, preferably one or two or more
cyclodextrins selected from .alpha.-cyclodextrin (6),
.beta.-cyclodextrin (7), .gamma.-cyclodextrin (8),
.delta.-cyclodextrin (9), .epsilon.cyclodextrin (10),
.zeta.-cyclodextrin (11), .eta.-cyclodextrin (12), and
.theta.-cyclodextrin (13), especially .alpha.-cyclodextrin,
.beta.-cyclodextrin or .gamma.-cyclodextrin; or primarily a
cyclodextrin derivative or mixture of cyclodextrin derivatives
selected from derivatives of a cyclodextrin, in which one or more
up to all of the hydroxy groups are etherified to an alkyl ether,
especially lower alkyl, such as methyl or ethyl ether, also propyl
or butyl ether; an aryl-hydroxyalkyl ether, such as
phenyl-hydroxy-lower-alkyl, especially phenyl-hydroxyethyl ether; a
hydroxyalkyl ether, in particular hydroxy-lower-alkyl ether,
especially 2-hydroxyethyl, hydroxypropyl such as 2-hydroxypropyl or
hydroxybutyl such as 2-hydroxybutyl ether; a carboxyalkyl ether, in
particular carboxy-lower-alkyl ether, especially carboxymethyl or
carboxyethyl ether; a derivatised carboxyalkyl ether, in particular
a derivatised carboxy-lower-alkyl ether in which the derivatised
carboxy is aminocarbonyl, mono- or di-lower-alkyl-aminocarbonyl,
morpholino-, piperidino-, pyrrolidino- or piperazinocarbonyl, or
alkyloxycarbonyl, in particular lower alkoxycarbonyl, such as
preferably a lower alkoxycarbonyl-lower-alkyl ether, for example
methyloxycarbonylpropyl ether or ethyloxy-carbonylpropyl ether; a
sulfoalkyl ether, in particular sulfo-lower-alkyl ether, especially
sulfobutyl ether; a cyclodextrin in which one or more OH groups are
etherified with a radical of formula
wherein alk is alkyl, especially lower alkyl, and n is a whole
number from 2 to 12, especially 2 to 5, in particular 2 or 3; a
cyclodextrin in which one or more OH groups are etherified with a
radical of formula ##STR4##
wherein
R' is hydrogen, hydroxy, --O--(alk--O).sub.z --H,
--O--(Alk(--R)--O--).sub.p --H or
--O--(alk(--R)--O--).sub.q --alk--CO--Y; alk in all cases is alkyl,
especially lower alkyl; m, n, p, q and z are a whole number from 1
to 12, preferably 1 to 5, in particular 1 to 3; and Y is OR.sub.1
or NR.sub.2 R.sub.3, wherein R.sub.1, R.sub.2 and R.sub.3
independently of one another, are hydrogen or lower alkyl, or
R.sub.2 and R.sub.3 combined together with the linking nitrogen
signify morpholino, piperidino, pyrrolidino or piperazino;
or a branched cyclodextrin, in which etherifications or acetals
with other sugars are present, and which are selected from
glucosyl-, diglucosyl-(G.sub.2 -.beta.-cyclodextrin), maltosyl- or
di-maltosyl-cyclodextrin, or N-acetylglucosaminyl-, glucosaminyl-,
N-acetylgalactosaminyl- and galactosaminyl-cyclodextrin; or a lower
alkanoyl, such as acetyl ester of a cyclodextrin; and
b) comprises a step for separating the epothilones, especially
epothilones A and B, from one another, which is characterised by
chromatography on a reversed-phase column with an eluant containing
a lower alkylcyanide, the chromatography being carried out on a
column material, especially an RP-18 material, which is charged
with hydrocarbon chains containing 18 carbon atoms, and employing
an eluant containing a lower alkyinitrile, especially acetonitrile,
in particular a mixture of lower alkylnitrile/water, preferably a
mixture of acetonitrile/water, preferably in a ratio of lower
alkylnitrile to water of ca. 1:99 to 99:1, primarily 1:9 to 9:1,
e.g. between 2:8 and 7:3, e.g. 3:7 or 4:6, whereby if desired, it
is possible to use further steps for working up and
purification.
The invention also relates in particular to a mutant derived from
the strain Sorangium cellulosum Soce90, especially a strain of
Sorangium cellulosum which is obtainable by mutagenesis, preferably
by one or more UV-induced mutagenesis steps (in particular by UV
radiation in the range 200 to 400, especially 250 to 300 nm) with
subsequent searching in each step for mutants having increased
epothilone production (in particular increased epothilone
concentration in the culture medium), this strain under otherwise
identical conditions producing more epothilones, in particular more
epothilone A and/or B, especially epothilone B, than Sorangium
cellulosum Soce90, especially the Sorangium cellulosum strain BCE
33/10, in particular BCE 114.
The invention relates in particular to the individual process steps
named in the examples or any combination thereof, the culture media
named therein, crystal forms and the strain described therein.
The invention also relates to new crystal forms of epothilone B,
especially a crystal form of epothilone B described as modification
B and in particular described as modification A.
The crystal forms can be distinguished in particular by their X-ray
diagrams. X-ray diagrams taken with a diffractometer and using
Cu-K.alpha..sub.1 -radiation are preferably used to characterize
solids of organic compounds. X-ray diffraction diagrams are used
particularly successfully to determine the crystal modification of
a substance. To characterize the existing crystal modification A
and crystal modification B of epothilone B, the measurements are
made at an angle range (2.theta.) of 2.degree. and 35.degree. with
samples of substance that are kept at room temperature.
The X-ray diffraction diagram thus determined (reflection lines and
intensities of the most important lines) from crystal modification
A (modification A) of epothilone B is characterized by the
following table.
2.theta. Intensity 7.7 very strong 10.6 weak 13.6 average 14.4
average 15.5 average 16.4 weak 16.8 weak 17.1 weak 17.3 weak 17.7
weak 18.5 weak 20.7 strong 21.2 strong 21.9 weak 22.4 weak 23.3
strong 25.9 average 31.2 weak 32.0 average
The invention also relates in particular to a new crystal form of
epothilones B, which is characterised by a melting point of more
than 120.degree. C., especially between 120.degree. C. and
128.degree. C., in particular 124-125.degree. C. Surprisingly, this
value is considerably higher than the values previously described
in literature. The invention relates especially to a crystal form
of epothilone B, which is characterised by the X-ray diffraction
diagram of the crystal form A and a melting point of above
120.degree. C., especially between 120.degree. C. and 128.degree.
C., for example between 124.degree. C. and 125.degree. C.
The X-ray diffraction diagram thus determined (reflection lines and
intensities of the most important lines) of crystal modification B
(modification B) of epothilone B is characterized by the following
table.
2.theta. Intensity 6.9 very strong 8.0 weak 8.3 average 10.8 strong
11.5 average 12.4 weak 13.1 strong 15.5 weak 16.2 weak 16.7 average
18.1 average 18.6 average 20.4 weak 20.9 strong 21.3 weak 21.5 very
weak 22.5 average 24.2 weak 25.1 average
The invention also relates in particular to a new crystal form of
epothilones B, which is characterised by a melting point of more
than 120.degree. C., especially between 124 and 125.degree. C.
Surprisingly, this value is considerably higher than the values
previously described in literature.
The new crystal forms are especially stable, particularly crystal
form A, and they are therefore suitable as active ingredients for
solid forms of administration, for storing in solid form or as
intermediates (with particularly good storability) in the
preparation of solid or liquid forms of administration.
The invention also relates to the use of the new crystal forms,
especially crystal form B, but primarily crystal form A (all
referred to hereinafter as active ingredient) in the production of
pharmaceutical preparations, new pharmaceutical preparations which
contain these new crystal forms, and/or the use of these compounds
in the treatment of proliferative diseases, such as tumours. In the
following, where pharmaceutical preparations or compositions which
comprise or contain the active ingredient are mentioned, in the
case of liquid compositions or compositions which no longer contain
the crystal form as such, this is always understood to mean also
the pharmaceutical preparations obtainable using the crystal forms
(for example infusion solutions obtained using crystal forms A or B
of epothilone B), even if they no longer contain the respective
crystal form (for example because they exist in solution).
The invention also relates especially to the use of a new crystal
form of epothilone B, especially the crystal form B or in
particular crystal form A, in the production of pharmaceutical
preparations, characterised by mixing a new crystal form of
epothilone B with one or more carriers.
The invention also relates to a method of treating warm-blooded
animals suffering from a proliferative disease, characterised by
administering a dose of epothilone B which is effective for
treating said disease in one or the new crystal forms to a
warm-blooded animal requiring such treatment, also including in
particular the treatment with those preparations that are produced
using one of the new crystal forms.
To produce the pharmaceutical preparations, the active ingredient
may be used for example in such a way that the pharmaceutical
preparations contain an effective amount of the active ingredient
together or in a mixture with a significant amount of one or more
organic or inorganic, liquid or solid, pharmaceutically acceptable
carriers.
The invention also relates to a pharmaceutical composition which is
suitable for administration to a warm-blooded animal, especially
humans, in the treatment of a proliferative disease, such as a
tumour, the composition containing an amount of active ingredient
that is suitable for treating said disease, together with a
pharmaceutically acceptable carrier.
The pharmaceutical compositions according to the invention are
those intended for enteral, especially nasal, rectal or oral, or
preferably parenteral, especially intramuscular or intravenous
administration to warm-blooded animals, especially humans, and they
contain an effective dose of the active ingredient on its own or
together with a significant amount of a pharmaceutically acceptable
carrier. The dose of the active ingredient is dependent on the type
of warm-blooded animal, the body weight, the age and the individual
condition, individual pharmacokinetic situations, the disease to be
treated and the type of administration.
The pharmaceutical compositions contain ca. 0.0001% to ca. 95%,
preferably 0.001% to 10% or 20% to ca. 90% of active ingredient.
Pharmaceutical compositions according to the invention may be
present for example in unit dose forms, such as in the form of
ampoules, vials, suppositories, dragees, tablets or capsules.
The pharmaceutical compositions according to the present invention
are produced by known processes, for example by conventional
dissolving, lyophilizing, mixing, granulating or manufacturing
processes.
Solutions of the active ingredient, also suspensions, and in
particular aqueous solutions or suspensions, are preferably
employed, whereby it is also possible, for example in the case of
lyophilised compositions which contain the active ingredient on its
own or together with a pharmaceutically acceptable carrier, for
example mannitol, for the solutions or suspensions to be prepared
prior to administration. The pharmaceutical compositions may be
sterilised and/or may contain excipients, for example
preservatives, stabilisers, moisture-retaining agents and/or
emulsion-forming agents, dissolving aids, salts for regulating
osmotic pressure and/or buffers, and they are produced by known
processes, for example by conventional dissolving or lyophilising
processes. The solutions or suspensions mentioned may comprise
viscosity-increasing substances, such as sodium
carboxymethylcellulose, carboxymethylcellulose, dextran,
polyvinylpyrrolidone or gelatin.
Suspensions in oil contain as the oil component vegetable oils,
synthetic oils or semi-synthetic oils, which are customary for
injection purposes. Notable examples are in particular liquid fatty
acid esters, which contain as the acid component a long-chained
fatty acid with 8 to 22, especially 12 to 22, carbon atoms, for
example lauric acid, tridecylic acid, myristic acid, pentadecylic
acid, palmitic acid, margaric acid, stearic acid, arachidic acid,
behenic acid or corresponding unsaturated acids, for example oleic
acid, elaidic acid, erucic acid, brassidic acid or linoleic acid,
if desired with the addition of antioxidants, for example vitamin
E, .beta.-carotene or 3,5-di-tert-butyl-4-hydroxytoluene. The
alcoholic component of these fatty acid esters preferably has a
maximum of 6 carbon atoms and is a mono- or polyhydroxy alcohol,
for example a mono-, di- or tri-hydroxy alcohol, for example
methanol, ethanol, propanol, butanol or pentanol, or an isomer
thereof, but especially glycol and glycerol. The following examples
of fatty acid esters may be mentioned in particular: propyl
myristate, isopropyl palmitate, "Labrafil M 2375" (polyoxyethylene
glycerol trioleate, Gattefosse, Paris), "Miglyol 812" (triglyceride
of saturated fatty acids having a chain length of 8 to 12 carbon
atoms, Huls AG, Germany), but in particular vegetable oils such as
cottonseed oil, almond oil, olive oil, castor oil, sesame oil,
soybean oil and in particular peanut oil.
The injection or infusion preparations are produced according to
customary methods under sterile conditions; the same applies also
to the filling of the compositions into ampoules or vials and
sealed containers.
Preference is given to an infusion solution which contains the
active ingredient and a pharmaceutically acceptable organic
solvent.
The pharmaceutically acceptable organic solvents which may be used
in a formulation according to the invention can be selected from
all such solvents which are familiar to a person skilled in the
art. The solvent is preferably selected from an alcohol, e.g.
absolute ethanol, ethanol/water mixtures, preferably 70% ethanol,
polyethylene glycol 300, polyethylene glycol 400, polypropylene
glycol and N-methylpyrrolidone, especially polypropylene glycol or
70% ethanol.
Particular preference is given to a formulation in pure
polyethylene glycol, which is diluted prior to infusion in an
appropriate solution, such as physiological saline.
The active ingredient is present in the formulation in a
concentration of 0.001 to 100 mg/ml, preferably from ca. 0.05 to 5
mg/ml, or from 5 to 50 mg/ml.
Formulations of this type are easily stored as vials or ampoules.
The vials or ampoules are typically made of glass, e.g. boron
silicate. The vials or ampoules may be appropriate for any volume
which is known from the prior art. They are preferably of
sufficient size to be able to accept 0.5 to 5 ml of the
formulation.
Prior to administration, the formulations have to be diluted in an
aqueous medium suitable for intravenous administration before the
active ingredient can be administered to patients.
It is preferable for the infusion solution to have the same or
basically the same osmotic pressure as body fluids. Consequently,
the aqueous medium contains an isotonic agent which has the effect
of rendering the osmotic pressure of the infusion solution the same
or basically the same as the osmotic pressure of body fluids.
The isotonic agent can be selected from all agents that are
familiar to a person skilled in the art, for example mannitol,
dextrose, glucose and sodium chloride. The isotonic agent is
preferably glucose or sodium chloride. The isotonic agents may be
used in quantities which impart the same or basically the same
osmotic pressure to the infusion solution as body fluids. The exact
quantities required can be determined by routine experiments and
depend on the composition of the infusion solution and the type of
isotonic agent.
The concentration of isotonizing agent in the aqueous medium
depends on the type of each agent used. If glucose is used, it is
preferably used in a concentration of 1 to 5% w/v, preferably 5%
w/v. If the isotonizing agent is sodium chloride, it is preferably
used in quantities of up to 1%, preferably ca. 0.9% w/v.
The infusion solution can be diluted with the aqueous medium. The
amount of aqueous medium used is chosen according to the desired
concentration of active ingredient in the infusion solution. The
infusion solution is preferably produced by mixing a vial or an
ampoule containing the infusion concentrate (see above) with an
aqueous medium, so that a volume of between 200 ml and 1000 ml is
attained with the aqueous medium. Infusion solutions may contain
other additives that are normally used in formulations for
intravenous administration. These additives also include
antioxidants.
Antioxidants may be used to protect the active ingredient from
degradation by oxidation. Antioxidants may be selected from those
which are familiar to the person skilled in the art and which are
suitable for intravenous formulations. The amount of antioxidant
can be determined by routine experiments. As an alternative to
adding an antioxidant, or additionally thereto, the antioxidant
effect can be achieved by restricting the oxygen (air) contact with
the infusion solution. This can be achieved in a simple way, by
treating the vessel containing the infusion solution with an inert
gas, e.g. nitrogen or argon.
Infusion solutions can be produced by mixing an ampoule or a vial
with the aqueous medium, e.g. a 5% glucose solution in WFI in an
appropriate container, e.g. an infusion bag or an infusion
bottle.
Containers for the infusion solutions may be selected from
conventional containers that are non-reactive with the infusion
solution. Among those suitable are glass containers, especially of
boron silicate, but plastic containers such as plastic infusion
bags, are preferred.
Plastic containers may also be made of thermoplastic polymers. The
plastic materials may also contain additives, e.g. softeners,
fillers, antioxidants, antistatic agents or other customary
additives.
Suitable plastics for the present invention should be resistant to
elevated temperatures used for sterilisation. Preferred plastic
infusion bags are the PVC materials which are known to the person
skilled in the art.
A large range of container sizes may be considered. When selecting
the size of the container, the factors to be taken into
consideration are especially the solubility of epothilones in an
aqueous medium, easy handling, and if appropriate, storage of the
container. It is preferable to use containers which hold between
ca. 200 and 1000 ml of infusion solution.
Owing to their good formulating properties, the new crystal forms
of epothilone B according to the invention are especially suitable
for the simple and reproducible production of the said infusion
solutions. However, the new crystal forms are especially suitable
for the production of pharmaceutical formulations which contain the
active ingredient in solid form, for example oral formulations.
Pharmaceutical formulations for oral application may be obtained by
combining the active ingredient with solid carriers, if desired by
granulating the resultant mixture, and further processing the
mixture, if desired or if necessary, after adding suitable
adjuvants, into tablets, dragee cores or capsules. It is also
possible to embed them in plastic substrates which enable the
active ingredient to be diffused or released in measured
quantities.
Suitable pharmaceutically employable carriers are especially
fillers, such as lactose, saccharose, mannitol or sorbitol,
cellulose preparations, and/or calcium phosphates, for example
tricalcium phosphate or calcium hydrogen phosphate, and binders,
such as starches, for example maize, wheat, rice or potato starch,
gelatin, tragacanth, methyl cellulose, hydroxypropyl methyl
cellulose, sodium carboxymethylcellulose, and/or polyvinyl
pyrrolidone, and/or, if desired, disintegrators, such as the
above-mentioned starches, crosslinked vinylpyrrolidones, agar,
alginic acid or a salt thereof, such as sodium alginate. Adjuvants
are in particular flow-improving agents and lubricants, e.g.
silicates, talcum, stearic acid or salts thereof, such as magnesium
or calcium stearate and/or polyethylene glycol. Dragee cores are
provided, if desired, with appropriate gastric-juice-resistant
coatings, using inter alia concentrated sugar solutions, gum
arabic, talcum, polyvinyl pyrrolidone, polyethylene glycol and/or
titanium dioxide, or coating solutions in suitable organic
solvents, or in order to produce gastric-juice-resistant coatings,
solutions of appropriate cellulose preparations, such as ethyl
cellulose phthalate or hydroxypropyl methyl cellulose phthalate.
Capsules are dry capsules consisting of gelatin or pectin, and if
required, a softener such as glycerol or sorbitol. The dry capsules
may contain the active ingredient in the form of granules, for
example with fillers, such as lactose, binders, such as starches,
and/or lubricants, such as talc or magnesium stearate, and where
appropriate stabilizers. In soft capsules, the active ingredient
may be present in dissolved or preferably suspended form, whereby
oily adjuvants such as fat oils, paraffin oil or liquid propylene
glycols are added; stabilizers and/or antibacterial additives may
also be added. Dyes or pigments can be added to the tablets or
dragee coatings, for example to improve identification or to
distinguish different dosages of active ingredient.
The usage in the treatment of a proliferative disease with one of
the crystal forms B and in particular A preferably takes place
whereby the crystal form (preferably as for the usage in the
preparation of an infusion solution, as described above) is
administered to a warm-blooded animal, especially a human, in a
dose which can be determined at between 20 and 133%, preferably
between 25 and 100%, of the Maximum Tolerated Dose (MTD) by
standard methods, for example using a modified Fibronacci series,
in which the increases in dosages for successive amounts are 100%,
67%, 50% and 40% followed by 33% for all subsequent doses; and, if
necessary, one or more further doses administered in the dosage
range given above for the first dose, each dose after a period of
time which allows sufficient recovery of the individual being
treated after the preceding administration, in particular one week
or more after the first administration, preferably 2 to 10 weeks,
especially 3 to 6 weeks after each preceding administration. In
general, this treatment scheme, in which a high dosage is
administered once, twice or several times with sufficiently long
intervals between the individual administrations for recovery to
take place, is preferred over a more frequent treatment with lower
doses, since hospitalisation is less frequent and for a shorter
period and an improved anti-tumour effect can be expected. The
dosage of epothilone B for humans is preferably between 0.1 and 50
mg/m.sup.2, preferably between 0.2 and 10 mg/m.sup.2.
The following Examples serve to illustrate the invention without
limiting its scope.
Caution: When handling epothilones, appropriate protective measures
must be taken, where necessary, in view of their high toxicity.
The 750 and 5000 liter fermenters used in the following are refined
steel fermenters from the company Alpha AG, Nidau, Switzerland.
EXAMPLE 1
Preparation of the Strain BCE33/10 and the Strain BCE63/114 By
Means of Mutation and Selection
The strain employed is the mutant BCE33/10 (deposited at the German
Collection of Microorganisms and Cell Cultures under number DSM
11999 on Feb. 9, 1998) or the mutant BCE63/114 (deposited at the
German Collection of Microorganisms and Cell Cultures under number
DSM 12539 on Nov. 27, 1998), which is derived from the strain
Sorangium cellulosum Soce90 by mutation and selection as described
below. In liquid media, the mutant BCE33/10, as well as BCE63/114,
forms bacilli typical of Sorangia, with rounded ends and a length
of 3-6 .mu.m, as well as a width of ca. 1 .mu.m. Sorangium
cellulosum Soce90 was obtained from the German Collection of
Microorganisms under number DSM 6773.
Preparation of the mutant BCE33/10 comprises three mutation steps
with UV light and selections of individual colonies. The procedure
in detail is carried out in accordance with the following operating
steps.
Cultivation From the Ampoule
The cells of the DSM6773 ampoule are transferred to 10 ml of G52
medium in a 50 ml Erlenmeyer flask and incubated for 6 days in an
agitator at 30.degree. C. and at 180 rpm. 5 ml of this culture are
transferred to 50 ml of G52 medium (in a 200 ml Erlenmeyer flask)
and incubated at 180 rpm for 3 days in an agitator at 30.degree.
C.
First UV Mutation Step and Selection
Portions of 0.1 ml of the above culture are plated out onto several
Petri dishes containing agar medium S42. The plates are then each
exposed to UV light (maximum radiation range of 250-300 nm) for 90
or 120 seconds at 500 .mu.watt per cm.sup.2. The plates are then
incubated for 7-9 days at 30.degree. C., until individual colonies
of 1-2 mm are obtained. The cells of 100-150 colonies are then each
plated out from an individual colony by means of a plastic loop in
sectors onto Petri dishes containing S42 agar (4 sectors per plate)
and incubated for 7 days at 300.degree. C. The cells that have
grown on an area of ca. 1 cm.sup.2 agar surface are transferred by
a plastic loop to 10 ml of G52 medium in a 50 ml Erlenmeyer flask
and incubated for 7 days at 180 rpm in an agitator at 30.degree. C.
5 ml of this culture are transferred to 50 ml of G52 medium (in a
200 ml Erlenmeyer flask) and incubated at 180 rpm for 3 days in an
agitator at 30.degree. C. 10 ml of this culture are transferred to
50 ml of 23B3 medium and incubated for 7 days at 180 rpm in an
agitator at 30.degree. C.
To determine the amounts of epothilone A and epothilone B formed in
this culture, the following procedure is followed. The 50 ml
culture solution is filtered through a nylon sieve (150 .mu.m pore
size), and the polystyrene resin Amberlite XAD16 retained on the
sieve is rinsed with a little water and subsequently added together
with the filter to a 50 ml centrifuge tube (Falcon Labware, Becton
Dickinson AG Immengasse 7, 4056 Basle). 10 ml of isopropanol
(>99%) are added to the tube with the filter. Afterwards, the
well-sealed tube is shaken for 1 hour at 180 rpm in order dissolve
the epothilone A and B, which is bonded to the resin, in the
isopropanol. Subsequently, 1.5 ml of the liquid is centrifuged, and
ca. 0.8 ml of the supernatant is added using a pipette to a HPLC
tube. The HPLC analysis of these samples is effected as described
below under HPLC analysis in the product analysis section. The HPLC
analysis determines which culture contains the highest content of
epothilone B. From the above-described sector plate of the
corresponding colony (the plates have been stored at 4.degree. C.
in the meantime), cells from ca. 1 cm.sup.2 of agar area are
transferred by a plastic loop to 10 ml of G52 medium in a 50 ml
Erlenmeyer flask and are incubated for 7 days at 180 rpm in an
agitator at 30.degree. C. 5 ml of this culture are transferred to
50 ml of G52 medium (in a 200 ml Erlenmeyer flask) and incubated at
180 rpm for 3 days in an agitator at 30.degree. C.
Second and Third UV Mutation Step and Selection
The procedure is exactly the same as described above for the first
UV mutation step, whereby the selected culture of the best colony
from the first UV mutation is used for the second mutagenesis. For
the third mutagenesis, the culture of the best colony from the
second mutagenesis is used accordingly. The best colony after this
third cycle of UV mutation steps, followed by selection of the
resulting strains for improved epothilone B production, corresponds
to mutant BCE33/10.
The strain BCE 63/114 is obtained from another (fourth) mutation
step from the strain BCE33/10, which is carried out in exactly the
same way as the above-mentioned mutation steps.
Preservation of the Strain
10 ml of a 3 day old culture in G52 medium (50 ml medium in a 200
ml Erlenmeyer flask, 30.degree. C. and 180 rpm) are transferred to
50 ml of 23B3 medium (in a 200 ml Erlenmeyer flask) and incubated
for 3 days at 180 rpm in an agitator at 30.degree. C. 1 ml portions
of this culture are removed in a form which is as homogeneous as
possible (prior to each removal the culture is shaken by hand in
the Erlenmeyer flask) together with the polystyrene resin Amberlite
XAD16 (polystyrene adsorption resin, Rohm & Haas, Frankfurt,
Germany), then filled into 1.8 ml Nunc cryotubes (A/S Nunc, DK 4000
Roslide, Denmark) and stored either at -70.degree. C. or in liquid
nitrogen.
Cultivation of the strains from these ampoules is effected by
heating them in the air to room temperature, and subsequently
transferring the entire content of the cryotube to 10 ml G52 medium
in an 50 ml Erlenmeyer flask and incubating for 5-7 days at 180 rpm
in an agitator at 30.degree. C.
Media
G52 Medium: yeast extract, low in salt 2 g/l (Springer, Maison
Alfort, France) MgSO.sub.4 (7 H.sub.2 O) 1 g/l CaCl.sub.2 (2
H.sub.2 O) 1 g/l soya meal defatted (Mucedola S.r.I., 2 g/l Settimo
Milan, Italy) potato starch Noredux (Blattmann, 8 g/l Wadenswil,
Switzerland) glucose anhydrous 2 g/l Fe-EDTA 8 g/l (Product No.
03625, Fluka 1 ml/l Chemie AG, CH) pH 7.4, corrected with KOH
Sterilisation: 20 mins. 120.degree. C.
S42 Agar-Medium
As described S. Jaoua et al. Plasmid 28, 157-165 (1992)
23B3 Medium: glucose 2 g/l potato starch Noredux (Blattmann, 20 g/l
Wadenswil, Switzerland) soya meal defatted (Mucedola S.r.I., 16 g/l
Settimo Milan, Italy) Fe-EDTA (Product No. 03625, Fluka, 0.008 g/l
Buchs, Switzerland) HEPES Fluka, Buchs, Switzerland 5 g/l
polystyrene resin XAD16 (Rohm and Haas) 2% v/v H.sub.2 O deionised
correction of pH to 7.8 with NaOH sterilisation for 20 mins. at
120.degree. C.
(HEPES=4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid)
EXAMPLE 2
Cultivation in Order to Produce the Epothilones Strain
Sorangium cellulosum Soce-90 BCE 33/10
(Example 1)
Preservation of the Strain
In liquid N.sub.2, as in Example 1.
Media: Precultures and intermediate cultures: G52 Main culture:
1B12 G52 Medium: yeast extract, low in salt (BioSpringer, 2 g/l
Maison Alfort, France) MgSO.sub.4 (7 H.sub.2 O) 1 g/l CaCl.sub.2 (2
H.sub.2 O) 1 g/l soya meal defatted Soyamine 50T (Lucas 2 g/l
Meyer, Hamburg, Germany) potato starch Noredux A-150 (Blattmann, 8
g/l Waedenswil, Switzerland) glucose anhydrous 2 g/l
EDTA-Fe(III)-Na salt (8 g/l) 1 ml/l pH 7.4, corrected with KOH
Sterilisation: 20 mins. 120.degree. C. 1B12 Medium: potato starch
Noredux A-150 (Blattmann, 20 g/l Waedenswil, Switzerland) soya meal
defatted Soyamine 50T (Lucas 11 g/l Meyer, Hamburg, Germany)
EDTA-Fe(III)-Na salt 8 mg/l pH 7.8, corrected with KOH
Sterilisation: 20 mins. 120.degree. C. Addition of cyclodextrins
and cyclodextrin derivatives: Cyclodextrins (Fluka, Buchs,
Switzerland, or Wacker Chemie, Munich, Germany) in different
concentrations are sterilised separately and added to the 1B12
medium prior to seeding.
Cultivation
1 ml of the suspension of Sorangium cellulosum Soce-90 BCE 33/10
from a liquid N.sub.2 ampoule is transferred to 10 ml of G52 medium
(in a 50 ml Erlenmeyer flask) and incubated for 3 days at 180 rpm
in an agitator at 30.degree. C., 25 mm displacement. 5 ml of this
culture is added to 45 ml of G52 medium (in a 200 ml Erlenmeyer
flask) and incubated for 3 days at 180 rpm in an agitator at
30.degree. C., 25 mm displacement. 50 ml of this culture is then
added to 450 ml of G52 medium (in a 2 liter Erlenmeyer flask) and
incubated for 3 days at 180 rpm in an agitator at 30.degree. C., 50
mm displacement.
Maintenance Culture
The culture is overseeded every 3-4 days, by adding 50 ml of
culture to 450 ml of G52 medium (in a 2 liter Erlenmeyer flask).
All experiments and fermentations are carried out by starting with
this maintenance culture.
Tests in a Flask
(I) Preculture in an Agitating Flask
Starting with the 500 ml of maintenance culture, 1.times.450 ml of
G52 medium are seeded with 50 ml of the maintenance culture and
incubated for 4 days at 180 rpm in an agitator at 30.degree. C., 50
mm displacement.
(ii) Main Culture in the Agitating Flask
40 ml of 1B12 medium plus 5 g/l 4-morpholine-propane-sulfonic acid
(=MOPS) powder (in a 200 ml Erlenmeyer flask) are mixed with 5 ml
of a 10.times. concentrated cyclodextrin solution, seeded with 10
ml of preculture and incubated for 5 days at 180 rpm in an agitator
at 30.degree. C., 50 mm displacement.
Fermentation
Fermentations are carried out on a scale of 10 liters, 100 liters
and 500 liters. 20 liter and 100 liter fermentations serve as an
intermediate culture step. Whereas the precultures and intermediate
cultures are seeded as the maintenance culture 10% (v/v), the main
cultures are seeded with 20% (v/v) of the intermediate culture.
Important: In contrast to the agitating cultures, the ingredients
of the media for the fermentation are calculated on the final
culture volume including the inoculum. If, for example, 18 liters
of medium+2 liters of inoculum are combined, then substances for 20
liters are weighed in, but are only mixed with 18 liters!
Preculture in an Agitating Flask
Starting with the 500 ml maintenance culture, 4.times.450 ml of G52
medium (in a 2 liter Erlenmeyer flask) are each seeded with 50 ml
thereof, and incubated for 4 days at 180 rpm in an agitator at
30.degree. C., 50 mm displacement.
Intermediate Culture, 20 Liters or 100 Liters
20 liters: 18 liters of G52 medium in a fermenter having a total
volume of 30 liters are seeded with 2 liters of the preculture.
Cultivation lasts for 3-4 days, and the conditions are: 30.degree.
C., 250 rpm, 0.5 liters air per liter liquid per min, 0.5 bars
excess pressure, no pH control.
100 liters: 90 liters of G52 medium in a fermenter having a total
volume of 150 liters are seeded with 10 liters of the 20 liter
intermediate culture. Cultivation lasts for 3-4 days, and the
conditions are: 30.degree. C., 150 rpm, 0.5 liters of air per liter
liquid per min, 0.5 bars excess pressure, no pH control.
Main Culture, 10 Liters, 100 Liters or 500 Liters
10 liters: The media substances for 10 liters of 1B12 medium are
sterilised in 7 liters of water, then 1 liter of a sterile 10%
2-(hydroxypropyl)-0-cyclodextrin solution are added, and seeded
with 2 liters of a 20 liter intermediate culture. The duration of
the main culture is 6-7 days, and the conditions are: 30.degree.
C., 250 rpm, 0.5 liters of air per liter of liquid per min, 0.5
bars excess pressure, pH control with H.sub.2 SO.sub.4 /KOH to pH
7.6+/-0.5 (i.e. no control between pH 7.1 and 8.1).
100 liters: The media substances for 100 liters of 1B12 medium are
sterilised in 70 liters of water, then 10 liters of a sterile 10%
2-(hydroxypropyl)-.beta.-cyclodextrin solution are added, and
seeded with 20 liters of a 20 liter intermediate culture. The
duration of the main culture is 6-7 days, and the conditions are:
30.degree. C., 200 rpm, 0.5 liters air per liter liquid per min.,
0.5 bars excess pressure, pH control with H.sub.2 SO.sub.4 KOH to
pH 7.6+/-0.5. The chain of seeding for a 100 liter fermentation is
shown schematically as follows: ##STR5##
500 liters: The media substances for 500 liters of 1B12 medium are
sterilised in 350 liters of water, then 50 liters of a sterile 10%
2-(hydroxypropyl)-.beta.-cyclodextrin solution are added, and
seeded with 100 liters of a 100 liter intermediate culture. The
duration of the main culture is 6-7 days, and the conditions are:
30.degree. C., 120 rpm, 0.5 liters air per liter liquid per min.,
0.5 bars excess pressure, pH control with H.sub.2 SO.sub.4 /KOH to
pH 7.6+/-0.5.
Product Analysis
Preparation of the Sample
50 ml samples are mixed with 2 ml of polystyrene resin Amberlite
XAD16 (Rohm+Haas, Frankfurt, Germany) and shaken at 180 rpm for one
hour at 30.degree. C. The resin is subsequently filtered using a
150 .mu.m nylon sieve, washed with a little water and then added
together with the filter to a 15 ml Nunc tube.
Elution of the Product From the Resin
10 ml of isopropanol (>99%) are added to the tube with the
filter and the resin. Afterwards, the sealed tube is shaken for 30
minutes at room temperature on a Rota-Mixer (Labinco BV,
Netherlands). Then, 2 ml of the liquid are centrifuged off and the
supernatant is added using a pipette to HPLC tubes.
HPLC analysis: Column: Waters-Symetry C18, 100 .times. 4 mm, 3.5
.mu.m WAT066220 + preliminary column 3.9 .times. 20 mm WAT054225
Solvents: A: 0.02% phosphoric acid B: Acetonitrile (HPLC-Quality)
Gradient: 41% B from 0 to 7 min. 100% B from 7.2 to 7.8 min. 41% B
from 8 to 12 min. Oven temp.: 30.degree. C. Detection: 250 nm,
UV-DAD detection Injection vol.: 10 .mu.l Retention time: Epo A:
4.30 min Epo B: 5.38 min
EXAMPLE 2A
Effect of the Addition of Cyclodextrin and Cyclodextrin Derivatives
to the Epothilone Concentrations Attained
All the cyclodextrin derivatives tested here come from the company
Fluka, Buchs, CH. The tests are carried out in 200 ml agitating
flasks with 50 ml culture volume. As controls, flasks with adsorber
resin Amberlite XAD-16 (Rohm & Haas, Frankfurt, Germany) and
without any adsorber addition are used. After incubation for 5
days, the following epothilone titres can be determined by
HPLC:
TABLE 1 Conc Epo Epo order [% w/ A B Addition No. v].sup.1 [mg/l]
[mg/l] Amberlite XAD-16 (v/v) 2.0 9.2 3.8 (% v/v)
2-hydroxypropyl-.beta.-cyclodextrin 56332 0.1 2.7 1.7
2-hydroxypropyl-.beta.-cyclodextrin " 0.5 4.7 3.3
2-hydroxypropyl-.beta.-cyclodextrin " 1.0 4.7 3.4
2-hydroxypropyl-.beta.-cyclodextrin " 2.0 4.7 4.1
2-hydroxypropyl-.beta.-cyclodextrin " 5.0 1.7 0.5
2-hydroxypropyl-.alpha.-cyclodextrin 56330 0.5 1.2 1.2
2-hydroxypropyl-.alpha.-cyclodextrin " 1.0 1.2 1.2
2-hydroxypropyl-.alpha.-cyclodextrin " 5.0 2.5 2.3
.beta.-cyclodextrin 28707 0.1 1.6 1.3 .beta.-cyclodextrin " 0.5 3.6
2.5 .beta.-cyclodextrin " 1.0 4.8 3.7 .beta.-cyclodextrin " 2.0 4.8
2.9 .beta.-cyclodextrin " 5.0 1.1 0.4 methyl-.beta.-cyclodextrin
66292 0.5 0.8 <0.3 methyl-.beta.-cyclodextrin " 1.0 <0.3
<0.3 methyl-.beta.-cyclodextrin " 2.0 <0.3 <0.3 2,6
di-o-methyl-.beta.-cyclodextrin 39915 1.0 <0.3 <0.3
2-hydroxypropyl-.gamma.-cyclodextrin 56334 0.1 0.3 <0.3
2-hydroxypropyl-.gamma.-cyclodextrin " 0.5 0.9 0.8
2-hydroxypropyl-.gamma.-cyclodextrin " 1.0 1.1 0.7
2-hydroxypropyl-.gamma.-cyclodextrin " 2.0 2.6 0.7
2-hydroxypropyl-.gamma.-cyclodextrin " 5.0 5.0 1.1 no addition 0.5
0.5 .sup.1) Apart from Amberlite (% v/v), all percentages are by
weight (% w/v).
Few of the cyclodextrins tested
(2,6-di-o-methyl-.beta.-cyclodextrin, methyl-.beta.-cyclodextrin)
display no effect or a negative effect on epothilone production at
the concentrations used. 1-2% 2-hydroxy-propyl-.beta.-cyclodextrin
and .beta.-cyclodextrin increase epothilone production in the
examples by 6 to 8 times compared with production using no
cyclodextrins.
EXAMPLE 2B
10 Liter Fermentation With 1%
2-(hydroxypropyl)-.beta.-cyclodextrin)
Fermentation is carried out in a 15 liter glass fermenter. The
medium contains 10 g/l of 2-(hydroxypropyl)-.beta.-cyclodextrin
from Wacker Chemie, Munich, Germany. The progress of fermentation
is illustrated in Table 2. Fermentation is ended after 6 days and
working up takes place.
TABLE 2 Progress of a 10 liter fermentation duration of culture [d]
Epo A [mg/l] Epo B [mg/l] 0 0 0 1 0 0 2 0.5 0.3 3 1.8 2.5 4 3.0 5.1
5 3.7 5.9 6 3.6 5.7
EXAMPLE 2C
100 Liter Fermentation With 1%
2-(hydroxypropyl)-.beta.-cyclodextrin)
Fermentation is carried out in a 150 liter fermenter. The medium
contains 10 g/l of 2-(hydroxypropyl)-.beta.-cyclodextrin. The
progress of fermentation is illustrated in Table 3. The
fermentation is harvested after 7 days and worked up.
TABLE 3 Progress of a 100 liter fermentation duration of culture
[d] Epo A [mg/l] Epo B [mg/l] 0 0 0 1 0 0 2 0.3 0 3 0.9 1.1 4 1.5
2.3 5 1.6 3.3 6 1.8 3.7 7 1.8 3.5
EXAMPLE 2D
500 Liter Fermentation With 1%
2-(hydroxypropyl)-.beta.-cyclodextrin)
Fermentation is carried out in a 750 liter fermenter. The medium
contains 10 g/l of 2-(hydroxypropyl)-.beta.-cyclodextrin. The
progress of fermentation is illustrated in Table 4. The
fermentation is harvested after 7 days and worked up.
TABLE 4 Progress of a 500 liter fermentation duration of culture
[d] Epo A [mg/l] Epo B [mg/l] 0 0 0 1 0 0 2 0 0 3 0.6 0.6 4 1.7 2.2
5 3.1 4.5 6 3.1 5.1
EXAMPLE 2E
Comparison Example 10 Liter Fermentation Without Adding an
Adsorber
Fermentation is carried out in a 15 liter glass fermenter. The
medium does not contain any cyclodextrin or other adsorber. The
progress of fermentation is illustrated in Table 5. The
fermentation is not harvested and worked up.
TABLE 5 Progress of a 10 liter fermentation without adsorber.
duration of Epothilone A Epothilone B culture [d] [mg/l] [mg/l] 0 0
0 1 0 0 2 0 0 3 0 0 4 0.7 0.7 5 0.7 1.0 6 0.8 1.3
EXAMPLE 3
Working Up of the Epothilones: Isolation From a 500 Liter Main
Culture
The volume of harvest from the 500 liter main culture of example 2D
is 450 liters and is separated using a Westfalia clarifying
separator Type SA-20-06 (rpm=6500) into the liquid phase
(centrifugate+rinsing water=650 liters) and solid phase (cells=ca.
15 kg). The main part of the epothilones are found in the
centrifugate. The centrifuged cell pulp contains <15% of the
determined epothilone portion and is not further processed. The 650
liter centrifugate is then placed in a 4000 liter stirring vessel,
mixed with 10 liters of Amberlite XAD-16 (Centrifugate:resin
volume=65:1) and stirred. After a period of contact of ca. 2 hours,
the resin is centrifuged away in a Heine overflow centrifuge
(basket content 40 liters; rpm=2800). The resin is discharged from
the centrifuge and washed with 10-15 liters of deionised water.
Desorption is effected by stirring the resin twice, each time in
portions with 30 liters of isoprapopanol in 30 liter glass stirring
vessels for 30 minutes. Separation of the isopropanol phase from
the resin takes place using a suction filter. The isopropanol is
then removed from the combined isopropanol phases by adding 15-20
liters of water in a vacuum-operated circulating evaporator
(Schmid-Verdampfer) and the resulting water phase of ca. 10 liters
is extracted 3.times. each time with 10 liters of ethyl acetate.
Extraction is effected in 30 liters glass stirring vessels. The
ethyl acetate extract is concentrated to 3-5 liters in a
vacuum-operated circulating evaporator (Schmid-Verdampfer) and
afterwards concentrated to dryness in a rotary evaporator (Buchi
type) under vacuum. The result is an ethyl acetate extract of 50.2
g. The ethyl acetate extract is dissolved in 500 ml of methanol,
the insoluble portions filtered off using a folded filter, and the
solution added to a 10 kg Sephadex LH 20 column (Pharmacia,
Uppsala, Sweden) (column diameter 20 cm, filling level ca. 1.2 m).
Elution is effected with methanol as eluant. Epothilone A and B is
present predominantly in fractions 21-23 (at a fraction size of 1
liter). These fractions are concentrated to dryness in a vacuum on
a rotary evaporator (total weight 9.0 g). These Sephadex peak
fraction (9.0 g) are thereafter dissolved in 92 ml of
acetonitrile:water:methylene chloride=50:40:2, the solution
filtered through a folded filter and added to a RP column
(equipment Prepbar 200, Merck; 2.0 kg LiChrospher RP-18 Merck,
grain size 12.mu.m, column diameter 10 cm, filling level 42 cm;
Merck, Darmstadt, Germany). Elution is effected with
acetonitrile:water=3:7 (flow rate=500 ml/min.; retention time of
epothilone A=ca. 51-59 mins.; retention time of epothilone B=ca.
60-69 mins.). Fractionation is monitored with a UV detector at 250
nm. The fractions are concentrated to dryness under vacuum on a
Buchi-Rotavapor rotary evaporator. The weight of the epothilone A
peak fraction is 700 mg, and according to HPLC (external standard)
it has a content of 75.1%. That of the epothilone B peak fraction
is 1980 mg, and the content according to HPLC (external standard)
is 86.6%. Finally, the epothilone A fraction (700 mg) is
crystallised from 5 ml of ethyl acetate:toluene=2:3, and yields 170
mg of epothilone A pure crystallisate [content according to HLPC (%
of area)=94.3%]. Crystallisation of the epothilone B fraction (1980
mg) is effected from 18 ml of methanol and yields 1440 mg of
epothilone B pure crystallisate [content according to HPLC (%) of
area=99.2%]. m.p. (Epothilone B): 124-125.degree. C.; .sup.1 H-NMR
data for Epothilone B: 500 MHz-NMR, solvent: DMSO-d6. Chemical
displacement .delta. in ppm relative to TMS. s=singlet; d=doublet;
m=multiplet
.delta. (Multiplicity) Integral (number of H) 7.34 (s) 1 6.50 (s) 1
5.28 (d) 1 5.08 (d) 1 4.46 (d) 1 4.08 (m) 1 3.47 (m) 1 3.11 (m) 1
2.83 (dd) 1 2.64 (s) 3 2.36 (m) 2 2.09 (s) 3 2.04 (m) 1 1.83 (m) 1
1.61 (m) 1 1.47-1.24 (m) 4 1.18 (s) 6 1.13 (m) 2 1.06 (d) 3 0.89 (d
+ s, overlapping) 6 .SIGMA. = 41
In this example (Example 3), epothilone B is obtained in the
crystal modification A, which is characterised by the X-ray
diffraction diagram of modification A (see general part of the
present disclosure).
EXAMPLE 4
Crystal Modification B of Epothilone B
50 mg of epothilone B (obtained for example as above) are suspended
in 1 ml of isopropanol and shaken for 24 hours at 25.degree. C. The
product is filtered and dried. After drying under a high vacuum,
epothilones B are obtained in the form of white crystals. The
crystal modification of the product is characterised by the X-ray
diffraction diagram of modification B (see general part of the
present disclosure).
EXAMPLE 5
3000 Liter Fermentation With
2-(hydroxypropyl)-.beta.-cyclodextrin)
Fermentation is carried out with the strain BCE 63/114 in a 5000
liter fermenter in 1B12 medium (filled volume 3000 liters).
Maintenance culture: Preparation is effected as described in
Example 1 (strain preservation) and 2 for the strain BCE33/10, but
using instead the strain BCE63/114.
Precultures
Preparation of the precultures is effected analogously to Example 2
(ii), but with the following precultures and with strain BCE
63/114:
From a 500 ml maintenance culture [as described in example 2 (ii)],
50 ml portions are placed in 4 Erlenmeyer flasks, thus producing
four 500 ml precultures in G52 medium for 3 days at 30.degree. C.
and at 180 rpm. These 4 precultures (2 liters) are then used for
three intermediate cultures each of 20 liters (G52 medium, 4 days,
30.degree. C., 250 rpm). 5 liter portions of these intermediate
cultures are used to produce three 50 liter intermediate cultures
(G52 medium, 3 days, 30.degree. C., 200 rpm). 50 liters of these 50
liter intermediate cultures are used twice to grow two 600 liter
intermediate cultures (G52 medium, 4 days, 30.degree. C., 120
rpm).
Main Culture
The media substances for 3000 liters are sterilised in 2100 liters
of water, then 300 liters of a sterile 10%
2-(hydroxypropyl)-.beta.-cyclodextrin solution are added, and
seeded with 600 liters of an intermediate culture. The duration of
the main culture is 6-7 days, and the conditions are: 30.degree.
C., 100 rpm, 0.5 liters of air per liter liquid per minute, 0.5
bars excess pressure, pH control with H.sub.2 SO.sub.4 /KOH to pH
7.6+/-0.5 (i.e. no control between pH 7.1 and 8.1). The progress of
fermentation is illustrated in Table 6.
TABLE 6 Progress of a 3000 liter fermentation duration of
Epothilone A Epothilone B culture [d] [mg/l] [mg/l] 0 0 0 1 0 0 2 0
0 3 2.1 1.6 4 4.1 2.9 5 5.2 3.8 6 5.5 4.3
Working Up and Isolation of the Epothilones From a 3000 Liter Main
Culture
(I) Resin Binding, Desorption and Extraction of the Epothilones
(Ethyl Acetate Extract)
The volume of harvest from the 3000 liter main culture is 2900
liters and is separated using a Westfalia clarifying separator Type
SA-20-06 (rpm=6500, flow rate 1400 liters/hour) into the liquid
phase (centrifugate+rinsing water=2750 liters) and solid phase
(cells=ca. 260 kg). The main part of the epothilones are found in
the centrifugate. The centrifuged cell pulp contains <15% of the
determined epothilone portion and is not further processed. The
2750 liter centrifugate is then placed in a 4000 liter steel
stirring vessel, mixed with 60 liters of Amberlite SAD-16
(centrifugate:resin volume=46:1) and stirred. After a period of
contact of 16-20 hours, the resin is centrifuged away in a Heine
overflow centrifuge (basket content 40 liters; rpm=2800). The
centrifuge is emptied by rinsing the basket content with deionised
water when the centrifuge is stationary. The XAD-16/deionised water
slurry is thereafter freed from water on a suction filter (.O
slashed.50 cm) and the resin washed with 30 liters of deionised
water. Desorption of the resin is effected by stirring it in a 1600
liter stirring vessel twice, each time with 220 liters of
isopropanol for 30 minutes. Separation of the isopropanol phase
from the resin takes place using a suction filter (.O slashed.50
cm). The isopropanol is then removed from the isopropanol phase by
adding 240-260 liters of water in a vacuum-operated circulating
evaporator (Schmid-Verdampfer) and the resulting water phase of ca.
125 liters is extracted 3.times. each time with 100-125 liters of
ethyl acetate. Extraction is effected in 1600 liter steel stirring
vessels. The ethyl acetate extracts are combined, concentrated to
3-5 liters in vacuum-operated circulating evaporators (Buchi
Verdampfer/Schmid-Verdampfer) and afterwards concentrated to
dryness in a rotary evaporator (Buchi type) under vacuum. An ethyl
acetate extract of 590 g is obtained.
HPLC Purification of the Ethyl Acetate Extract (Separation of
Epothilones A and B)
Ca. 300 g of the above-mentioned ethyl acetate extract (with a
content of ca. 1-1.5% epothilone B) are suspended in 1.5 liters of
acetonitrile/water=3/1 (v/v), the solution is filtered through a
folded filter and the filtrate added to a C-18 RP column [Prochrom
apparatus with 30 cm internal column diameter (Prochrom,
Champigneulles, France) 25 kg YMC gel, ODS-A, 120 Angstroem pore
diameter, 5-15 .mu.m grain size, spherical]. Elution is effected
with acetonitrile/water=4/6 (v/v) at a flow rate of 2300 ml/min.
Fractionation is monitored by means of on-line HPLC [rapid HPLC at
high temperatures (ca. 80.degree. C.), on a short separating column
(4.6 mm internal diameter.times.75 mm length) and very small RP-18
particles (3.5 .mu.m spherical), typical analysis times are <1
minute, detection at 250 nm]. The valuable fractions (those with
only epothilone B) are combined, the acetonitrile removed by
distillation and the aqueous phase extracted twice with isopropyl
acetate. The organic phases are concentrated by distillation and
the isopropyl acetate extract is obtained as the residue of
evaporation.
(iii) Silica Gel Filtration of the Isopropyl Acetate Extract
Ca. 10 g of the combined residues of evaporation thus obtained
(with a content of ca. 23% of epothilone B) are dissolved at room
temperature in 360 ml of ethyl acetate, the solution is filtered
through a folded filter and added to a column of silica gel
(Prochrom apparatus with 10 cm internal column diameter, 1.5 kg ICN
18-32 .mu.m). Elution is effected with ethyl acetate/n-hexane=4/1
(v/v) at a flow rate of 250 ml/min at room temperature.
Fractionation is monitored with a UV detector at 250 nm. The
valuable fractions are combined, the solvent removed by
distillation and concentrated to dryness.
(iv) Extraction of the Pure Epothilones
Ca. 48 g of the combined residues obtained under (iii) (with a
content of ca. 90% of epothilone B) are dissolved in 1150 ml of
methanol, mixed with 14.5 g of activated carbon and subsequently
filtered through a folded filter. The clear filtrate is
subsequently concentrated to dryness and the residue is
recrystallised from 317 ml of methanol. 29.5 g of epothilone B are
obtained in a purity of 99.7%, and with a melting point of
124.degree. C.
EXAMPLE 6
Infusion Concentrate
By dissolving in polyethylene glycol PEG 300, crystal modification
A of epothilone B, or crystal modification B of epothilone B, is
produced in a preconcentrate to produce infusion solutions, and
stored in vials.
* * * * *